Kidney and Body Fluids: Proceedings of the 28th International Congress of Physiological Sciences, Budapest, 1980

possible.

NEURAL CONTROL OF RENAL FUNCTION*

Carl W. Gottschalk**, Romulo E. Colindres, Nicholas G. Moss, Paula R. Rogenes and Laszlo Szalay***,     Departments of Medicine and Physiology University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA

Publisher Summary

This chapter discusses that efferent renal nerve stimulation affect both proximal tubular transport of salt and water and the resistance of the renal vessels. It highlights the possibility of a neural mechanism, a renorenal reflex, being responsible for the coordination of the excretory activity of the two kidneys that occurs when the function of one of the kidneys is altered. The condition under which the mechanism becomes operative requires further identification, but it is not restricted to the anesthetized state. Renal chemoreceptors and the mechanoreceptors provide an afferent mechanism for ipsilateral and contralateral renorenal reflexes integrated at the spinal cord level. This appears to be the operative mechanism in at least one instance of coordination of renal excretory activity. The full extent of the neural mechanism, involving different neurotransmitters with opposing effects, almost certainly exceeds in complexity of the simple system.

This morning I wish to consider the role of the renal nerves in maintaining the homeostasis of the body fluids. I will discuss the efferent control of excretory activity and the nature of the renal receptors that send afferent impulses to the neural axis when stimulated. I will also consider the possibility of a neural mechanism, a renorenal reflex, being responsible for the coordination of the excretory activity of the two kidneys that occurs when the function of one of the kidneys is altered.

Claude Bernard (1859) was the first to report that acute section of the splanchnic nerves in an anesthetized dog results in an increase in urine production by the ipsilateral kidney. This observation has been repeatedly confirmed over the subsequent years. The two problems addressed by most investigators have been, (first) whether the effect is exclusively hemodynamic in origin due to an increase in glomerular filtration rate and renal blood flow, or whether there is also an effect on the tubular mechanisms for reabsorption, and secondly, whether the effect, whatever its mechanism, is evident only under the abnormal circumstances of anesthesia and surgery, and thus should not be considered a response to physiological alterations. Until recently the answer to the first question was that the effect was exclusively hemodynamic, but I will review the compelling evidence that the sympathetic efferent inflow to the kidney has a direct effect on tubular reabsorptive mechanisms which may under certain circumstances be magnified by hemodynamic factors, although the latter need not occur. I do not believe that the answer to the second question has yet been completely resolved. Almost all of the published evidence favors the view that it is apparent only when the animal is severely stressed. More recent studies, however, indicate that the effects of denervation may be seen in conscious animals.

The neural efferent mechanism for control of salt and water excretion has been intensively investigated in recent years in 3 laboratories, by Dr. Takacs and his colleagues at Semmelweis University here in Budapest, by Dr. DiBona and colleagues at the University of Iowa, and by my associates in the Chapel Hill Micropuncture Laboratory. The results obtained in these laboratories have been complementary.

The recent description of a tubular innervation provides an anatomical basis for the tubular functional effects reported by these 3 laboratories. It has been known since the studies of Bradford (1889) that there is a rich sympathetic innervation of the blood vessels of the kidney; although there have been periodic reports of a direct tubular innervation there have been at least as many reports denying this. In the early 1970s Barajas and Mueller presented for the first time convincing evidence for a direct innervation of the tubular cells in the cortex of monkey and rat kidneys (Barajas and Mueller 1973 and Mueller and Barajas 1972). Using electron microscopic and histochemical methods these investigators demonstrated adrenergic nerve terminals separated from proximal and distal tubular cells only by basement membrane material. This relationship is identical to that observed between similar vesiculated varicosities and vascular smooth muscle, a site where synaptic transmission is thought to occur.

MICROPUNCTURE AND CLEARANCE EXPERIMENTS

Although a proximal tubular effect was first reported by Bencsath et al. (1972), the Chapel Hill group was the first to provide extensive micropuncture documentation for such a result. Bello-Reuss et al. (1975) characterized the renal response to acute unilateral denervation in an extensive study of sham denervated and denervated kidneys. Denervation of a kidney was accomplished by stripping the renal artery of its adventitia and coating it with a solution of 10% phenol in alcohol. For a variety of reasons the functional changes observed cannot be attributed to a direct effect of phenol on kidney function: no norepinephrine could be detected in the kidneys several days after denervation; incomplete denervation from splanchnic nerve crushing produced similar, but quantitatively smaller results than the apparently complete chemical denervation, and these effects were reversed by electrical stimulation of the distal end of the cut splanchnic nerve. Coating the artery with lidocaine instead of phenol produced similar but transitory effects.

No changes were observed in any function of either kidney of sham-denervated rats. Following unilateral denervation in hydropenic animals urine volume from the denervated kidney increased to about twice its control value, and urinary sodium excretion increased sixfold. There was no change in urine volume or sodium excretion from the innervated kidney. Glomerular filtration rate (GFR) and renal plasma flow (RPF) remained unchanged in both kidneys after the procedure. SNGFR remained unchanged after denervation. The fluid-to-plasma ratio of inulin decreased in samples of fluid collected from late proximal tubules of denervated kidneys indicating a 60% decrease in water and sodium reabsorption by the proximal tubule. Absolute water and sodium reabsorption increased in the loop of Henle, distal convolution, and collecting ducts, but not enough to compensate for the 60% decrease in the proximal tubule.

Denervation caused no change in estimated glomerular capillary or efferent arteriolar pressure. There were very small increases in hydrostatic pressure in proximal and distal convolutions and in small peritubular capillaries. Since there was no change in whole kidney or single nephron GFR and renal plasma flow was unchanged, it is unlikely that there was a change in overall or superficial nephron colloid osmotic pressure or significant redistribution of renal blood flow. It thus appears that the physical factors did not play an important role in the observed changes in tubular reabsorption.

Similar results were observed in animals expanded 10% above their body weight by an infusion of isotonic saline solution (Bello-Reuss et al. 1977). There was no change in GFR or RPF in either kidney after unilateral denervation or sham denervation. Urine flow and sodium excretion by denervated kidneys was doubled. Simultaneously urine flow and sodium excretion by the contralateral innervated kidneys fell by half so that there was little change in total salt and water excretion. I will return to this striking finding shortly. After denervation SNGFR remained unchanged. The F/P inulin ratio in fluid from late proximal tubules decreased, indicating a fall in water and sodium reabsorption in the proximal tubule of more than 50%. Absolute water and sodium reabsorption increased after denervation in the loop of Henle, distal convolution and collecting ducts but not enough to prevent the natriuresis and diuresis.

In another series of experiments in anesthetized rats the natriuresis and diuresis resulting from unilateral crushing of the greater splanchnic nerve was reversed by electrical stimulation of the distal portion of the cut nerve with square wave pulses of 0.5 msec duration, voltage twice threshold, and 1 or 2 Hz frequency (Bello-Reuss et al. 1976). Kidney GFR and RPF and SNGFR remained unchanged during stimulation. Nerve stimulation produced a reduction of approximately 25% in urine flow and sodium excretion due to increased water reabsorption in the proximal tubule. In 5 of 6 animals, stimulation at 1 Hz was followed by an increase in proximal F/P inulin ratio. In the one animal in which there was no change in F/P inulin there was no change in sodium excretion by the kidney. For unknown reasons, the appropriate fibers apparently were not stimulated in that animal. On cessation of stimulation the F/P inulin ratio returned to control values. Subsequent stimulation at 2 Hz caused an increase in F/P inulin ratio in all animals. Recordings of the compound action potential of the stimulated nerves indicated that the effect on tubular reabsorption resulted from stimulation of slowly conducting C fibers.

Recent studies both in Budapest (Bencsath et al. 1979) and in Chapel Hill (Colindres et al. 1980) demonstrate that the denervation effect is not a transient one that is observed only immediately after denervation, since similar results were observed in rats and dogs when they were anesthetized weeks or months after unilateral denervation.

Szalay et al. (1977a, b, c, d) have studied other proximal tubular transport systems in chronically denervated kidneys. Very interestingly, all solute systems studied were affected. Glucose Tm and TmPAH were depressed, as were the tubular reabsorption of phosphate and urate. Thus it appears that there is substantial impairment of multiple proximal tubular active transport systems after renal denervation.

MECHANISM OF NEURAL EFFECT

None of these studies provides major insight into whether the neural effect on the tubular transport systems is direct (due to the neurotransmitter or electrical events at the nerve endings) or indirect (secondary to an effect of change in nerve traffic on the renin-angiotensin, prostaglandin, kallikrein, or other humoral systems). If a humoral substance is involved it does not appear to gain access to the general circulation, since a similar change in sodium excretion by opposite, innervated kidneys is not observed.

The experiments of DiBona and colleagues not only give convincing evidence of an effect of renal nerve stimulation on sodium excretion, but also provide useful information about the mechanism. They have demonstrated in the dog that direct low frequency electrical stimulation of the splanchnic nerve or reflex activation of high-pressure or low-pressure baroreceptors results in a change of sodium excretion without a change in the kidneys GFR or RPF; sodium excretion returned to control levels when activation was stopped (Prosnitz and DiBona 1978 and Zambraski et al. 1976a). Low frequency renal nerve stimulation did not alter the intrarenal distribution of blood flow as assessed by the radioactive microsphere technique (Slick et al. 1975). Very clearly then, the change in sodium excretion resulted from a change in tubular reabsorption without a change in intrarenal hemodynamics.

DiBona et al. have established the adrenergic specificity of the antinatriuretic response to low frequency electrical stimulation (Slick et al. 1975 and Zambraski et al. 1976b). In paired studies, phenoxybenzamine, an alpha-adrenergic receptor antagonist, or guanethidine, an adrenergic blocking agent, abolished a previously demonstrated antinatriuretic response to low frequency electrical stimulation.

Since renal nerve stimulation is known to cause release of renin and prostaglandins from the kidney, and since these substances might influence tubular sodium reabsorption, DiBona and colleagues have investigated whether the natriuresis resulting from nerve stimulation might be mediated by these humoral systems (DiBona et al. 1977 and Zambraski and DiBona 1976). There was no inhibition of the response to low frequency nerve stimulation following the administration of Sar-1, Ala-8, angiotensin II, a competitive angiotensin II antagonist, or indomethacin. Therefore the response does not appear to be mediated by the intrarenal action of circulating angiotensin II or prostaglandin.

Bencsath et al. (1972) have demonstrated that the neural effect on tubular reabsorption of sodium can be dissociated from those on renin release. Thus their studies also support the concept that denervation natriuresis does not result from a change in activity of the renin-angiotensin system.

Quite recently Bello-Reuss (1980) has reported that adding 10−6 M 1-norepinephrine to the bathing solution but not the perfusate reversibly stimulated fluid reabsorption by approximately two-thirds in isolated perfused segments of the proximal pars convoluta. Norepinephrine did not affect water flux in isolated segments of the pars recta. The addition of 10−4 M propranolol to the bath blocked the effect of norepinephrine on water flux in pars convoluta segments, suggesting that the increase of water flux is mediated by stimulation of beta adrenergic receptors. This stands in apparent conflict with DiBonas results who reported that, an alpha blocker, phenoxybenzamine abolished the effect of nerve stimulation. The dosage of propranolol employed by Bello-Reuss was high, and this may have affected its receptor specificity.

Pastoriza (1979) has studied the effects of the addition of 10−6 M norepinephrine or 10−6 M phentolamine, an alpha blocker, to the peritubular perfusate of in vivo perfused proximal convoluted tubules. Norepinephrine increased and phentolamine decreased fluid reabsorption by approximately one-third These experiments also demonstrate a direct tubular action of norepinephrine, presumably due to stimulation of alpha receptors located in the basolateral membrane.

Brenner and colleagues have studied the effects of catecholamines on isolated rabbit cortical collecting tubule fragments (Iino and Brenner 1979 and Iino et al. 1979). The addition of norepinephrine or isoproterenol to the bath led to a decrease in transtubular PD. The response to isoproterenol was prevented by prior addition to the bath of the beta adrenergic blocker, propranolol. The effect of isoproterenol on PD was due to an increase in chloride flux from lumen to bath, thereby increasing net chloride absorption. These studies are of particular interest since they indicate an effect of catecholamines on salt and water transport in another section of the nephron and participation of beta receptors.

Dopamine, a potent natriuretic catecholamine, may also be involved in the homeostatic control of salt and water balance. Gill (1969 and 1979), who has long supported a role of the renal nerves in the regulation of renal tubular sodium reabsorption, has recently reviewed the role of low pressure and high pressure baroreceptors in the control of renal efferent nerve activity and release of norepinephrine and possibly dopamine. Gill and colleagues have presented evidence that in humans an increase in dietary sodium intake or infusion of saline results in a decrease in urinary excretion of norepinephrine and an increase in dopamine excretion (Alexander et al. 1974). He has also reported that infusion of dibutyryl adenosine 3′,5′ cyclic monophosphate into a renal artery decreases proximal tubular reabsorption of sodium (Gill and Casper 1971a). Activation of proximal adenylate cyclase may be the mechanism for the decrease in proximal reabsorption associated with renal B-adrenergic activation (Gill and Casper 1971b).

STUDIES IN CONSCIOUS ANIMALS

After accepting that efferent sympathetic nerve stimulation of the kidney can affect both the proximal tubular transport system for salt and water as well as the renal vascular resistance, the question remains under what circumstances are these mechanisms activated, Certainly the success with kidney transplantation with initial total denervation argues that the neural mechanisms are not essential for overall kidney function and preservation of homeostasis. It does not follow however that the function of the transplanted kidney is normal; often it is not, although it is very difficult to ascertain the cause(s). Further, when only one kidney is present there is no need for coordination of activity of two kidneys, about which I will speak shortly.

The literature contains numerous studies of dogs with unilateral renal denervation. Although the results have been variable, the overall evidence points to a lack of significant difference in function of the denervated and innervated kidneys in the absence of anesthesia and surgery. Time does not permit a review of the relevant literature, but I do wish to call to your attention two recent publications, the one by Lifsohitz and the other by Sadowski and colleagues.

Lifschitz (1978) studied conscious dogs following unilateral renal denervation, confirmed by kidney norepinephrine analysis, with split bladders so that urine could be collected from each kidney separately. The effects of two protocols were investigated in the same dog; volume expansion to 5% of BW followed by a hemorrhage of 2% BW, and on another day the same procedures in reversed order. Urine volume, sodium and potassium excretion were all higher during volume expansion and lower during hemorrhage. There were also large changes in renal hemodynamics; GFR was consistently lower during hemorrhage than during volume expansion and renal plasma flow generally was lower. With both protocols however the responses of the denervated and intact kidneys were similar. Not surprisingly Dr. Lifschitz concluded that these results suggest that the renal nerves do not have a significant role in the regulation of sodium excretion in conscious animals.

I think that at least 2 other explanations are possible for the lack of a neural effect. These animals were subjected to great stress, and I believe it possible that their circulating catecholamine levels were sufficiently high to override the differences in neuronal catecholamine release between the two kidneys. Those of us who have pet dogs at home recognize the difference in heart rate, respiration and other behavior of a dog in the laboratory setting. Another possibility to explain the similarity of results from the two kidneys also involves catecholamine release with denervation supersensitivity such that similar effects were present in both denervated and innervated kidneys. To my knowledge there have been no studies on the tubular mechanism in regard to the possibility of denervation supersensitivity.

Sadowski et al. (1979) have recently reported results which lead them to warn against incautious extrapolation of the data from renal denervation experiments performed under anesthesia to physiological conditions. They studied a small number of dogs while conscious and anesthetized before and after saline loading. Barbiturate anesthesia distinctly depressed hemodynamics and increased the functional difference between denervated and innervated kidneys. In conscious moderately hydrated dogs the denervated kidney excreted slightly more sodium and water; after saline loading higher excretion was observed on the innervated side. The greater natriuretic response of the innervated kidney to saline infusion was believed to be due to inhibition of the sodium retaining action of renal efferent nerve activity by acute extracellular volume expansion. We agree with these investigators that the large differences observed in salt and water excretion in anesthetized unilaterally denervated animals results from an increase in sympathetic stimulation to the innervated kidney as well as the absence of neural impulses reaching the denervated kidney. But to repeat, the differences in function between innervated and denervated kidneys in the absence of anesthesia were small.

Rogenes has recently completed a study of kidney function in unanesthetized rats with chronic unilateral renal denervation in the Chapel Hill Micropuncture Laboratory. Seven to 9 days after unilateral denervation rats were anesthetized with ether and vascular and ureteral cannulas implanted. After allowing several hours for recovery, inulin and PAH clearances and salt and water excretion by the two kidneys were measured separately. These conscious rats were studied first in the euvolemic state and then following 3% extracellular fluid volume expansion. The rat was then anesthetized with pentobarbital and the observations repeated in the anesthetized volume expanded animal. Thus the same animals were studied while conscious and euvolemic, conscious and volume expanded and anesthetized volume expanded. Rats which had a sham-unilateral denervation operation 7–9 days before the clearance procedures were also studied. In the sham-operated animals the function of the two kidneys was quite similar during all three conditions. There was a fall in CIn and CPAH in the volume expanded animals following induction of anesthesia, but this did not reach statistical significance. There was no difference in the rate of urine flow or sodium excretion between the two kidneys in these sham-operated animals under any one of the 3 conditions.

In the 10 animals with chronic unilateral denervation and CPAH were significantly greater in the denervated kidneys during euvolemia, but not following volume expansion, although differences persisted. There was no change in CIn or CPAH following induction of anesthesia in the volume expanded state. In contrast to the sham-operated animals in all 3 conditions salt and water excretion by the denervated kidneys were greater than by the innervated kidneys. The difference did not reach statistical significance in the euvolemic state, but did in the conscious animals following volume expansion. After induction of anesthesia the differences in salt and water excretion by the 2 kidneys increased considerably. The increased difference was due to a fall in sodium excretion by the innervated kidneys plus increased excretion by the denervated kidneys. Thus the differential effect during anesthesia is due to a combination of increased sympathetic flow to the innervated kidneys as well as absence of sympathetic stimulation to the denervated kidneys. I wish to emphasize a finding of major interest. Total salt excretion by the two kidneys during volume expansion was the same in the conscious and anesthetized states even though the excretion of the denervated and innervated kidneys both changed. Was this merely fortuitious or did it result from a homeostatic coordination of excretory activity of the two kidneys by some mechanism, perhaps neural? More about this later.

While working this year in the Chapel Hill Micropuncture Laboratory Dr. Laszlo Szalay has also studied the excretory activity of conscious volume expanded rats with chronic unilateral denervation. He confirms Rogenes’ finding of increased salt and water excretion by denervated kidneys and has extended the results by demonstrating increased excretion of phosphate and calcium as well. Earlier he had reported decreased proximal phosphate reabsorption by the chronically denervated kidney of anesthetized dogs (Szalay et al. 1977b).

We recognize that the conditions under which Rogenes and Szalay made their observations on conscious rats were not normal. Nevertheless, the experiments do extend the conditions under which a neural effect has been observed and demonstrate that it is not restricted to unconscious animals.

COORDINATION OF EXCRETORY ACTIVITY OF THE TWO KIDNEYS

There are numerous reports that a change in excretory activity of one kidney is followed by a reciprocal change in excretory activity of the other kidney such that total excretion of salt and water by the animal is unchanged or minimally affected. Such a relation may occur following unilateral occlusion of the renal artery, vein or ureter and after unilateral denervation. There is very little information concerning the responsible mechanism and the same mechanism does not necessarily underlie all responses of this type. The immediate changes in excretion typically result from a change in tubular activity and are not dependent upon changes in hemodynamics, although such may occur and augment the changes in excretion of salt and water (Peters 1963). We have been interested in the possibility that these changes may be neurally mediated and represent a renorenal reflex. Experiments in Chapel Hill and Iowa City indicate that such is the case in at least one circumstance, the decrease in salt and water excretion by the innervated kidney following the diuresis and natriuresis produced by acute contralateral denervation in volume expanded rats.

Colindres and colleagues (1980) have made the following observations. As anticipated, acute unilateral denervation in anesthetized volume expanded rats produced an ipsilateral diuresis and natriuresis. There was a simultaneous decrease in sodium and water excreted by the contralateral innervated kidney such that total excretion was unchanged. Similar observations were made in rats in which 1 kidney had been denervated one to two weeks earlier. Acute denervation of the innervated kidney resulted in profuse ipsilateral diuresis and natriuresis. No change occurred, however, in salt and water excretion by the contralateral chronically denervated kidney.

In another 6 animals efferent nerve traffic to the contralateral kidney was measured after acute unilateral denervation. It increased to a mean of 45% above control levels at 0–30 min with a further significant increase to 66% above control levels 30-60 min after denervation. DiBona and Rios (1980) have reported very similar observations on efferent nerve traffic to the contralateral kidney following unilateral denervation.

It is known that the excretory changes produced by sympathetic nerve stimulation are precisely those seen in these responding kidneys and the results are interpreted to indicate that the functional excretory changes in an innervated kidney after contralateral acute renal denervation are caused by reflex increase in nerve traffic to the responding kidney.

It is possible that the increase in efferent traffic in the nerves to the innervated kidney is a result of the interruption of inhibitory afferent signals originating in the denervated kidney. If this interpretation is correct the electrophysiological and functional changes in the responding kidney constitute an example of an inhibitory renorenal reflex. Calaresu et al. (1978) have described electrophysiological characteristics of renorenal reflexes in the cat and Macfarlane (1970) has postulated the existence of a renorenal reflex to explain the vasoconstriction seen in one kidney of the dog after injection of acetylcholine into the contralateral renal artery. It is relevant to note that the kidney possesses both mechano- and chemoreceptors. It is probable therefore that the afferent limb of any postulated renorenal reflex involves impulses originating in either or both of these types of receptors.

The excretory changes of the innervated kidney after contralateral renal denervation were appropriate to maintain body fluid homeostasis. This could be interpreted to suggest a reflex control mechanism operating through the renal nerves to insure a balanced excretory output from the two kidneys. We believe however that this interpretation must be considered with great caution. Such an adaptive neural response would involve a) a primary change in excretory function in one kidney, b) detection of its altered function by receptors in that kidney, and c) changes in afferent impulses from that kidney which reflexly lead to a change in efferent impulses to the contralateral kidney regulating its excretory activity. In our experiments it was inevitable that the denervation procedure simultaneously interrupted ipsilaterally both afferent and efferent nerve traffic. Thus the denervation diuresis and the contralateral antidiuresis although occurring simultaneously could well have been independent phenomena and a cause and effect relationship should not be inferred.

The results of this study do suggest that alterations in afferent renal nerve activity might produce reflex enhancement or inhibition of contralateral nerve activity as required to balance excretory function of both kidneys. The renal receptors which initiate impulses capable of influencing the opposite kidney and the stimuli that trigger renorenal reflexes must be identified. Also the effects of afferent impulses on blood pressure and other cardiovascular functions deserve intensive study.

R1 AND R2 CHEMORECEPTORS

Like other visceral organs the kidneys have an abundant afferent (sensory) innervation and I would like to consider the nature of the renal receptors whose stimulation leads to impulses to the neural axis. In cat and dog experiments receptors have been described that are stimulated by increased arterial, venous or ureteral pressure and which are considered mechanoreceptors. Recordati, Moss and their collaborators working in Chapel Hill Micropuncture Laboratory have recently described in the rat two other groups of renal receptors sensitive to chemical alterations of their environment and which they have provisionally termed renal R1 and R2 chemoreceptors (Recordati et al. 1978 and 1980a). The R1 receptors are silent under control conditions and are activated by severe renal ischemia. The R2 receptors exhibit a resting discharge and respond to backflow into the renal pelvis of nondiuretic urine and other solutions of specific chemical composition.

Their studies were done in anesthetized, artificially ventilated male Sprague-Dawley rats. The distal portion of centrally cut nerves from the kidney were dissected for single unit recordings.

R1 chemoreceptors were activated by marked impairment of renal blood flow produced by clamping the renal artery, severe hypotension or prolonged occlusion of the renal vein, and by systemic asphyxia. The same units were not responsive to increases or decreases in systemic arterial pressure over a range of 40–190 mm Hg, to increases in ureteral pressure up to 50 mm Hg or to moderate changes in renal venous pressure. Thus they are not mechanoreceptors. Their pattern of activation during renal ischemia was characterized by trains of impulses, resulting from a change in their chemical environment during ischemia.

It appeared to be possible to wash out of the kidney the substance(s) responsible for the activation of these fibers during ischemia. When a clamped kidney was perfused with isotonic saline through a cannula inserted into the renal artery between the clamp and the kidney the activation disappeared. The neural activity reappeared promptly after the perfusion was stopped and while the renal artery was still clamped. The investigators were not able to identify the substance responsible for the change(s) in chemical environment which lead to activation. Such things as vasoactive hormones, adenosine, local accumulation of CO2, change of pH, and increase in interstitial potassium or increase in intracellular sodium are obvious possibilities.

More recently Recordati and colleagues (1980a) have described another set of receptors, their R2 units. These units are also responsive to change in their chemical environment, and it is likely that they have been confused with mechanoreceptors in the past. In these experiments a special catheter with multiple branches was placed into the ureter of one kidney to allow convenient perfusion of the pelvis with solutions of different composition.

The R2 units were active under control conditions and were markedly stimulated by backflow of nondiuretic urine into the renal pelvis. When isotonic saline was substituted for the nondiuretic urine there was no neural activation even though the increase in ureteral pressure was the same.

Single units sensitive to the backflow of nondiuretic urine were isolated and their responses to solutions of various compositions were tested: sodium chloride 850 mEq/l produced marked activation but lower concentrations produced little or none; 1 M mannitol produced activation after a slight delay, and 1 M urea appeared to be inhibitory. Potassium chloride solutions of 50, 100 and 150 mEq/l were all markedly excitatory. Similar concentrations of potassium chloride are usual in nondiuretic urine. In all of these experiments activation was dependent on the chemical nature of the test solution and was independent of the degree of increase in ureteral pressure.

Renal ischemia activated these fibers also, but R2 receptors are different from the R1s which only respond to renal ischemia. The latter are not spontaneously active and are not stimulated by backflow of nondiuretic urine into the pelvis.

Analogous to the results with R1 receptors, it was also possible to reduce the rate of firing of activated R2 receptors by perfusion of the kidney with isotonic saline. In recordings from multifiber preparations perfusion of the kidney with isotonic saline following activation by backflow of urine reduced the rate of firing to control levels; activation promptly reappeared following cessation of saline perfusion. In the same multifiber preparation it was possible to reduce the rate of firing produced by renal artery occlusion to background level by perfusion of the kidney with saline; again activity returned promptly following cessation of perfusion. And it was also possible to reduce the rate of firing to background levels during renal artery occlusion by vigorous backflow of isotonic saline into the renal pelvis. Apparently the receptors are located beneath and close to the pelvic epithelium and are readily accessible from either the pelvic or the tissue side.

The rate of spontaneous discharge of R2 receptors was markedly reduced during extracellular volume expansion suggesting an involvement in regulation of the extracellular fluid volume and composition. Thus the R2 receptors respond to changes in ionic concentration in the renal interstitium such as may be caused by urinary solutes crossing the pelvic epithelium, by alterations in renal blood flow or by renal functional demands. They do not respond nonspecifically to changes in total osmolality of fluid back perfused into the pelvis but demonstrate chemical specificity. They appear to be most sensitive to changes in potassium concentration. Obviously leakage of potassium from surrounding cells may be a natural stimulus.

It seems unlikely that the unique role of the R2 receptors is nociception. To the contrary, they can be activated during perfusion of the pelvis at low pressures and are unaffected by distension of the pelvis with isotonic saline. Further in nondiuretic conditions they have a high resting discharge rate which cannot be assumed to give rise to a painful sensation.

PARTICIPATION OF R2 CHEMORECEPTORS IN RENORENAL REFLEXES

Recordati in Milan and Rogenes in Chapel Hill have independently studied the effect of stimulation of R2 chemoreceptors by backflow of nondiuretic urine into the renal pelvis to determine if this leads to reflex activation of efferent renal nerve activity (ERNA). Both report positive results.

Recordati et al. (1980b) recorded ipsilaterally from multifiber preparations. They found that backflow of urine caused a 5–30% increase in ERNA in rats with intact nervous systems and denervated baroreceptors. In rats with the spinal cord cut at the Cl level, activation was greater and ranged from 40-80%. Thus they concluded that afferent nerve activity produces excitatory reflexes which are integrated at the spinal cord level. There appeared to be inhibition from higher neural centers.

Rogenes measured ERNA before, during and after backflow of nondiuretic urine into the pelvis of the ipsilateral or the contralateral kidney. She found an average activation of 20.9% and 21.5%, respectively, in multiunit recordings. Efferent renal nerve activity quickly returned to control values following removal of the stimulating urine. She also demonstrated that spinal cord section at a high cervical level did not prevent reflex activation.

Quite importantly Rogenes has made observations on single units following dissection of the nerve into a fine filament. During R2 receptor stimulation she found increased firing frequency ranging from 50–180% over control in some efferent units, no change in others and, less frequently, an actual reduction of activity in some units. These differences in single unit response provide direct evidence that the renal nerves contain a mixture of efferent fibers presumably reflecting functional heterogeneity.

Thus the studies by Recordati and Rogenes demonstrate that stimulation of the recently described R2 receptors leads to ipsilateral and contralateral renorenal reflexes which are integrated at the spinal cord level. They extend the earlier studies in which electrical stimulation of renal afferent fibers led to a change in efferent nerve activity (Aars and Akre 1970, Calaresu et al. 1978 and Ueda et al. 1967).

SUMMARY

Finally and very briefly to recapitulate. Efferent renal nerve stimulation can affect both proximal tubular transport of salt and water as well as the resistance of the renal vessels. The conditions under which the mechanism becomes operative requires further identification, but it is not restricted to the anesthetized state. The recently described renal chemoreceptors and the previously known mechanoreceptors provide an afferent mechanism for ipsilateral and contralateral renorenal reflexes integrated at the spinal cord level. This appears to be the operative mechanism in at least one instance of coordination of renal excretory activity. The full extent of the neural mechanism, probably involving different neurotransmitters with opposing effects, almost certainly exceeds in complexity the simple system so far described.

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*Supported by National Institutes of Health Grants HL02334 and NS1132 and by a Grant-in-Aid from the American Heart Association

**Career Investigator, American Heart Association

***Career Investigator Fellow, American Heart Association

A MEMBRANE-MOLECULAR APPROACH TO RENAL PHYSIOLOGY

Rolf Kinne,     Albert Einstein College of Medicine, New York, USA

Publisher Summary

This chapter describes the term membrane–molecular approach, which is used to isolate those areas of the plasma membrane of an epithelial cell that constitute the luminal or the antiluminal border of the cell in such a way that the transport function of the molecules present in the membranes remain intact and is studied in vitro. The main focus is placed on membrane events. The chapter discusses a new approach, which makes it possible (1) to distinguish between the role of the plasma membrane and the role of other cellular components in transcellular transport and regulation, (2) to localize the transport elements situated in the two opposite poles of the cell, namely, the luminal and contraluminal membranes, and (3) to characterize these transport elements in terms of driving forces, molecular properties, and sensitivity to regulatory influences. The chapter also explains that the membranes should be left essentially unaltered by the purification procedures so that the vesicles reflect very closely the functions of the intact cell in vivo.

1 INTRODUCTION

When I first learned that I had been elected for the first Robert Pitts Memorial Lectureship, I immediately recalled my first meeting with this eminent renal physiologist. It took place during a dinner at Dr. Ullrich’s home at Frankfurt and the conversation dealt to my surprise with modern architecture and the influence of the Bauhaus school on the functional design of large building complexes and of small items for daily use. This burning interest in the functional design of the whole electrolyte metabolism as well as in the functional design of detailed mechanisms is reflected in his main scientific contributions concerning acid base balance and intrarenal metabolism and last but not least, in his textbook. His thoughts were clear, his language was lucid and his experiments were like borderstones for a house, each of them performed in the awareness of its contribution to the goal of the investigation and to the whole gestalt of the problem (18,19).

My approach to renal physiology might stem from the same roots; if I would have to define it very briefly, it is the interest in the functional organization of epithelial cells in general and of the renal cells in particular. To phrase it differently: how is the epithelial cell organized to fulfill its main function, namely transcellular transport; what are the elements involved in it, what is their spatial arrangement, and what is the chain of events comprising transcellular transport?

At the time when I began to ask these questions, the transport function of the various segments of the nephron had been defined by clearance and micropuncture experiments, the nature of the elements involved in the transport processes, were, however, essentially unknown. Information about their properties could be derived only indirectly from the limited knowledge of salt and solute gradients assumed to exist across the two essential borders of the epithelial cell, the luminal border, facing the tubular fluid, and the antiluminal border facing the interstitial fluid that equilibrates with the blood in the capillaries.

The most direct way to obtain information pertinent to the above defined functional organization seemed the membrane-molecular approach to renal physiology which I am going to discuss today. The term membrane-molecular approach will be used in this lecture (as has been done at other occasions (11,12,13) in describing the attempt to isolate those areas of the plasma membrane of an epithelial cell that constitute the luminal or the antiluminal border of the cell in such a way, that the transport function of the molecules present in the membranes remain intact and can be studied in vitro. Thus renal physiology will be dealt with from a different viewpoint. The main focus will be placed on membrane events.

The work I am going to review today is the summary of experiments performed mainly during my stay in Frankfurt at the Max Planck Institut fur Biophysics in the Department of Physiology. I would like at this point to express my deep appreciation to Prof. Dr. K.J. Ullrich, the director of this department for his continuous generous support and guidance in my personal and scientific development.

2 THE FUNCTIONAL ORGANIZATION OF THE PROXIMAL TUBULAR CELL

2.1 Transcellular transport

The proximal tubular cell is a prime example for a highly differentiated and organized cell. The luminal brush border has a different morphology, stability and composition than the basal-lateral membranes forming the contraluminal labyrinth. This fact makes it possible to isolate the two membranes based on their difference in surface charge density by electrophoresis (10), by differential precipitation with divalent cations (2) or by density gradient centrifugation (15).

The main elements involved in active solute transfer across the proximal tubule found so far are ion-sensitive ATPases, on one hand, and sodium-cotransport systems on the other hand. The Na-K-ATPase generates and sustains the electrochemical potential difference for sodium across the luminal and contraluminal cell border. This enzyme is found only in the basal-lateral plasma membranes of the cell (10). Other ATP driven transport systems described in renal plasma membranes are an ATP dependent Ca-pump which has an apparent Km for Ca of 1µM and is found exclusively in the basal-lateral plasma membranes (9) (see fig. 1A) and an ATP driven proton pump in the brush border membranes (fig. 1B) (E. Kinne, R. Beauwens and R. Kinne, unpublished observations).

Figure 1A ATP-driven calcium uptake by basal-lateral plasma membrane vesicles isolated from rat kidney proximal tubule. The incubation medium contained 0.12 CaCl2, 100 mM KCl, 5mM MgCl2, and 20mM HEPES-Tris, pH 7.0, incubation temperature 25°C. Uptake of radioactive calcium was determined by a rapid filtration technique. ATP-concentration 5mM, A 23187 concentration 10–6M. Redrawn after Gmaj et al. (1979).

Figure 1B ATP-driven proton ejection from ATP-loaded brush border membrane vesicles isolated from rat kidney proximal tubule. The membranes were preloaded with ATP and a regenerating system, proton ejection was measured with a pH electrode. Incubation temperature 37°C, Concentration of CFCCP 5 g/mg protein. (Unpublished results from E. Kinne, R. Beuwens, R.