‘4 v , . H , x I l ‘ . .. ..‘ u‘ ‘ “a. M .. . . . I ‘ {J y '~.',I,';.‘ -. .u'." ‘; .‘.. ‘ . ‘.").'._‘_‘..’ “1}". ..1 ‘ r ‘ .; a‘ 4 " . ‘ . {J “-1“ ”v.3... .v. ‘ K «v ‘t'v ‘ . 'o ' . _‘ .7 . M . ' ‘ v , ‘.., .,:- "‘II‘>Q\V' .:..u V .....r,. a, " ‘ v "!" vi::;:_?(: ..\.4‘ ‘1' Y.‘ ‘ .... '1‘ c:::'.:.';'. ' '~- v"; .V ,, “.-'-.~‘- 'v" ....u ...~q nh‘nu. -‘ .w I _' . > . ”2'. ,..--..~...~. t” .-,.',H.'.V.:,.IF .. . ,— q ‘. .’. _ . . . .. . . .‘ , . ... 4 .. ‘..,,.".. .q..,..7, .. .1..-‘-.4.' '" V. J rd} ‘4. n. v‘.fl-V.‘.... ‘ <1 ' . . . . ~ ‘ .. . ‘ . - ~ .. .... . .‘ ~ ‘ ‘ . ..‘ r - . ‘ ‘l . ‘ ‘ . ..v . . ‘ ‘ . . . . ‘ . , . ‘ . ‘ ‘ . . ‘ Al , . ‘ . ‘. . . . . 1 . ., . . H . , ‘. . . . ' n I - v ~ llx ‘ ~ , ». ‘ . . . v \‘ CARDIOPULMONARY SYMPATHETIC AFFERENT INFLUENCES ON THE KIDNEY BY Linda Joy Macklem A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology 1978 A €,y‘\\43/\%> . ABSTRACT CARDIOPULMONARY SYMPATHETIC AFFERENT INFLUENCES ON THE KIDNEY by Linda J. Macklem Activation of cardiopulmonary sympathetic afferent nerves (CPSAN) electrically or by intravascular volume expansion can alter renal efferent nerve activity, al- though it is not known if this reflex requires the inte- grity of the brainstem. To determine if this reflex requires supraspinal pathways, experiments were conducted in vago- tomized, sino—aortic denervated cats prior to and following C1 spinal cord transection. Although the cardiopulmonary-renal reflex could be demonstrated prior to spinal transection, following tran— section this reflex could not be elicited either electrically or with intravascular volume expansion, indicating that this reflex requires supraspinal pathways. Since CPSAN stimulation can alter renal nerve aCtivity in intact cats, the influences of CPSAN on renal function was determined. Afferent stimulation at 1—2 trains per sec inhibited renal nerve activity and caused a diuresis and natriuresis. Afferent stimulation at 5-7 Hz had no net effect on renal nerve activity or renal function. Thus, CPSAN can influence renal nerve activity and renal sodium and water excretion, and therefore, may contribute to intra- vascular volume control. ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Lynne C. Weaver for her patient guidance throughout the course of this study. Her advice and encouragement have been invaluable. I would also like to thank Dr. Jerry B. Hook whose criti- cism of this project has been particularly helpful. Last, I would like to thank the members of my committee, Dr. S. Richard Heisey and Dr. Kenneth E. Moore. They have always been available when their assistance was needed. Finally, I would like to extend special appreciation to my parents, Mr. and Mrs. F.S. Macklem, for their enthus- iasm and emotional support throughout the past several years. It is to them that this thesis is dedicated. ii TABLE OF CONTENTS Page ACKNOWLEDGMENTS .......................................... 11 TABLE OF CONTENTS ....................................... iii LIST OF TABLES ........................................... .v LIST OF FIGURES .......................................... vi HISTORICAL REVIEW ......................................... 1 General Background ..................................... l The Volume Receptor Hypothesis: Reflex Control ........ l of Renal Electrolyte and Water Excretion Spinal Sympathetic Reflexes ...................... ' ...... 7 Effect of Renal Nerves on Renal Sodium and ............ 12 Water Excretion RATIONALE ................................................ 22 METHODS .................................................. 23 General Methods ....................................... 23 Specific Methods 1: Spinal Reflex Experiments ......... 25 A. Activation of Afferent Nerves....... ............ 25 1. Activation by Electrical Stimulation.. ....... 25 2. Activation by Intravascular Volume... ........ 25 Expansion B. Neural Recording ................................ 26 C. Protocol ........................................ 26 1. Responses to Activation of Afferent .......... 26 Nerves with Electrical Stimulation 2. Responses to Activation of Afferent..........28 Nerves with Intravascular Volume Expansion iii TABLE OF CONTENTS (CONT.) Page D. Data Analysis ................................... 29 1. Responses to Activation of Afferent Nerves. .29 with Electrical Stimulation 2. Responses to Activation of Afferent Nerves...3o with Intravascular Volume Expansion Specific Methods II: Renal Function Experiments ....... 31 A. Activation of Afferent Nerves ................... 31 B. Neural Recording ......... ...................... 32 C. Sample Collection for Estimation of Renal.......32 Function D. Protocol............. ..... . ........... .. ........ 33 B. Data Analysis......... .......................... 34 l. Neural Recordings ............................ 34 2. Renal Function ............................... 34 RESULTS .................................................. 41 I. Spinal Reflex Experiments .......................... 41 A. Responses to Activation of Afferent Nerves ...... 41 by Electrical Stimulation B. Responses to Activation of Afferent Nerves".... 44 by Intravascular Volume Expansion II. Renal Function Experiments ......................... 44 A. Renal Nerve Responses to Afferent Stimulation...44 B. Renal Function Responses to Afferent Stimu- ..... 45 lation DISCUSSION ............................................... 76 SUMMARY AND CONCLUSIONS .................................. 87 BIBLIOGRAPHY ............................................. 33 APPENDIX ................................................. viii iv LIST OF TABLES Table Page 1 Sympathetic reflex characteristics prior -------- 59 to and following C1 Spinal transection LIST OF FIGURES Figure Page 1 General methods used in determining the------o-38 existense of a spinal cardiopulmonary- renal reflex. 2 General methods used in determining the -------- 40 influence of cardiOpulmonary sympathetic afferent nerves on renal function. 3 Effect of cardiopulmonary sympathetic .......... 48 afferent nerve stimulation on renal sym- pathetic efferent nerve activity prior to spinal cord transection. 4 Excitatory response of a renal sympathetic ..... 50 efferent nerve to cardiOpulmonary sympa- thetic afferent nerve stimulation prior to and following spinal cord transection. 5 Effect of cardiopulmonary sympathetic .......... 52 afferent nerve stimulation on cardiac sympathetic efferent nerve activity prior to spinal cord transection. 6 Excitatory response of a cardiac sympa—........54 thetic efferent nerve to cardiopulmonary sympathetic afferent nerve stimulation prior to and following spinal cord tran- section. 1 7 Effect of renal sympathetic afferent nerve ..... 56 stimulation on renal sympathetic efferent nerve activity prior to spinal cord tran- section. 8 Excitatory responses of a renal sympathetic....58 efferent nerve to renal sympathetic affer- ent stimulation prior to and following spinal cord transection. vi LIST OF FIGURES (CONT.) Figure Page 9 Effects of intravascular volume expan- ......... 61 sion on renal nerve activity and central venous pressure (CVP) following Cl spinal cord transection. 10 Renal efferent nerve responses evoked by ........ 63 cardiopulmonary sympathetic afferent nerve stimulation at 1-2 trains/sec. 11 Effect of cardiopulmonary sympathetic ........... 65 afferent stimulation (1-2 trains/sec) on sodium excretion (U aV), potassium. excretion (UKV) and urine flow rate (V) in 7 cats. 12 Effect of cardiOpulmonary sympathetic ........... 67 afferent stimulation (1-2 trains/sec) on glomerular filtration rate (C ), mean arterial pressure (MAP), and frac¥ional excretion of sodium (FENa) in 7 cats. 13 Summary of renal responses to cardio---. -------- 69 pulmonary sympathetic afferent stimu— lation at l~2 trains/sec. in 7 cats. 14 Summary of renal responses to cardiopul-........7l monary sympathetic afferent stimulation at 5-7 H2 in 5 cats. 15 Effect of time on sodium excretion (U V) ....... 73 potassium excretion (UKV) and urine fIgw rate (V) in 6 cats. 16 ' Effect of time on glomerular filtration ......... 75 rate (C ), mean arterial pressure (MAP), and fraé¥ional excretion of sodium (FENa) in 6 cats. vii HISTORICAL REVIEW General Background: Autonomic control of the thoracic and abdominal viscera has been studied extensively. Yet, our knowledge is still incomplete. Until very recently, it was thought that afferent information concerning the cardio- vascular system was carried almost solely by nerves emanating from the carotid sinus and aortic arch, with a modest contri- bution made by other vagally innervated receptors. However, Malliani and coworkers have shown that vascular structures innervated by sympathetic afferent nerves can reflexly influence the cardiovascular system (61,65). Often these influences do not require an intact neuraxis (62,65) indicating that spinal control of the circulatory system may be significant. Very recently, it has been shown that cardiOpulmonary sympathetic afferent nerves can reflexly influence renal sympathetic efferent nerve activity (101). Since evidence has been accumulating that renal nerve discharge can alter renal function (27), afferent fibers travelling with sym- pathetic nerves may possibly alter renal function. This would in turn indicate an influence of the sympathetic afferent system in intravascular volume control. The Volume Receptor Hypothesis: Reflex Control of Renal Electrolyte and Water Excretion: In 1935, Peters suggested that one factor contributing to renal control of intra- vascular volume might involve the distribution of blood in the vascular tree (84). Since that time numerous studies have been conducted in an attempt to locate receptors in the vasculature which might influence the kidney. In 1954, Gauer g; a1. showed that exaggerated negative pressure breathing resulted in a diuresis in dogs (31). This procedure is believed to engorge the thoracic vasculature with blood. It was hypothesized that stretch receptors in the thoracic vasculature excited by the redistribution of fluid might form the afferent loop in a reflex resulting in diuresis. In 1956, Henry _t a1. found that distension of a small balloon placed in the left atrium also caused a diuresis (40). This procedure caused increases in pressure in the pulmonary circulation as well as in the left atrium. However, constriction of the pulmonary veins, causing the same elevation in pulmonary pressure as left atrial dis- tension, did not cause a diuresis. Since distension of the left atrium resulted in increased afferent activity in the cardiac branch of the vagus nerve, and vagal cooling blocked the diuretic response to distension, it was concluded that vagally innervated receptors in the left atrium and terminal pulmonary veins were likely the initiators of the diuretic reflex (40,41). Previously, anatomical evidence of such receptors had been provided by Nonidez (79). The diuresis resulting from negative pressure breathing had been assumed to be due to decreases in plasma concen- trations of antidiuretic hormone, as the diuresis had resulted partly from increases in free water clearance (16,31,98). With the use of balloon distension of the left atrium, workers from several laboratories were able to demonstrate decreases in plasma concentrations of antidiuretic hormone which corresponded to increases in left atrial pressure (2,43). Also, it was shown that the diuretic response to left atrial distension could be attenuated in some instances by infusion of antidiuretic hormone (55,58). Not only was there evidence that renal excretion of water might be partially mediated by vagally innervated receptors in the thoracic vasculature, but there were also indications that renal excretion of salt might be regulated by similar receptors. In 1956, Davis 3: 31. (25) had shown that constriction of the vena cava above the diaphragm stimulated aldosterone secretion. Mills gt a1. (73) con- firmed this finding and added that the decrease in aldo- sterone secretion seen on release of caval constriction could be blocked by vagotomy. Aldosterone has long been known to increase.sodium reabsorption by the kidney. Brennan 33 31. (17) later confirmed that antidiuretic hormone release could be inhibited by increases in left atrial pressure, and added that renin release could be inhibited by increases in right atrial pressure. Renin acts as an enzyme in the conversion of angiotensin I to angiotensin II, angiotensin II then causing secretion of aldosterone (24). Thus, vagally innervated receptors in the thoracic vasculature appear to be at least partially responsible for alterations in both antidiuretic hormone and aldosterone secretion, and therefore, could influence renal excretion of sodium and water through humoral mechanisms. In 1972, Karim gt a1. demonstrated that activation of vagally innervated left atrial receptors could alter renal sympathetic efferent nerve discharge (48). This study was particularly important in that it showed fractionation of the sympathetic response. That is, excitation of vagally innervated left atrial receptors caused an excitation of cardiac sympathetic efferent nerve discharge and an inhi- bition of renal sympathetic efferent discharge. More evidence of reflex vagal control of renal nerve activity was provided by Clement 33 31. (21), who demonstrated that volume expansion could inhibit and hemorrhage could enhance renal sympathetic nerve activity. That these changes in renal nerve activity could be eliminated or attenuated by vagal section again indicates a reflex alteration due to vagally innervated receptor activity. Sympathetic nerves also carry afferent fibers which innervate receptors in the thoracic vasculature. These receptors have been demonstrated anatomically (76,79,80,8l) and physiologically (19,20,65,100). By occluding the coro- nary arteries to produce myocardial ischemia, Brown demon- strated that activation of certain receptors in the coronary vasculature may be responsible for the sensation of cardiac pain (18). Malliani gt 31. (65) and Brown and Malliani (19) then showed that some sympathetically innervated receptcrrs in the coronary vasculature could respond to alterations in coronary arterial pressure and therefore appeared to be mechanoreceptors. The 'reflex response to pressure alter- ations was altered cardiac sympathetic efferent discharge. Ueda g; 31. were able to locate receptors sensitive to mechanical stimuli whose fibers travelled in sympathetic nerves (100). These workers found receptors in the main pulmonary artery, pulmonary veins, pleura, superior and inferior venae cavae, thoracic aorta, aortic root, left coronary artery, and all four chambers of the heart. However, most of the fibers examined by this group were not spontan- eously active. This would limit the amount of information received by the central nervous system from sympathetic afferent fibers and might indicate that these fibers serve to inform the central nervous system only of gross abber- ations in cardiovascular function. Several years following the study by Ueda §£_a13, workers in Malliani's laboratory demonstrated tonic sympathetic afferent activity stemming from receptors in the atria and ventricles (63,64). Activity from these fibers was consistently in phase with identi- fiable parts of the cardiac cycle, although a given fiber did not necessarily fire during each cycle. Fibers from the atria discharged with atrial systole, closure of the artio-ventricular valves, or atrial filling. Fibers from the ventricles discharged 40-120 msec following the onset of the Q wave. Because discharge of these fibers can be linked to normal cardiac events, it is possible that these sympathetic afferent fibers are involved in supplying the central nervous system with information about the normal cardiac cycle. Subsequently, tonic activity also was demonstrated in sympathetic afferent fibers from the pulmonary artery, pulmonary veins, and aorta (78,57,59) increasing the amount of information potentially conveyed to the central nervohs system by sympathetic afferent nerves. That electrical activation of these afferent nerves could reflexly alter arterial pressure (18,85), heart rate (61), and cardiac contractility (62) coupled with the knowledge that these nerves are tonically active, indicates that they may be important in regulation of normal cardiovascular function. Recently, it has been shown that electrical activation of cardiac sympathetic afferent fibers in cats in which cranial nerves IX and X have been severed also can alter renal sympathetic efferent nerve activity (101). High frequency stimulation of afferent fibers in the inferior cardiac nerve (a sympathetic nerve) increased, and low frequency stimulation decreased renal nerve activity. Also, in the same study, intravascular volume expansion caused an inhibition of renal nerve activity. This inhi— bition was not seen following section of dorsal roots T1 through T5. Sectioning these dorsal roots eliminates sympathetic afferent influences from the heart and lungs and, therefore, indicates that activation of sympathetically innervated mechanoreceptors in the thoracic vasculature may have been responsible for reflex inhibition of renal nerve activity. Previously, Gilmore and coworkers had shown that cardiac denervation could attenuate the natriuretic response to volume expansion only if cardiac afferent fibers travel- ing in sympathetic nerves were eliminated (35,72). These last studies suggest that sympathetic afferent fibers originating from receptors in the thoracic vasculature may contribute to renal control of intravascular volume by mechanisms other than systemic arterial pressure altera- tion. Spinal Sympathetic Reflexes: In 1969, Malliani, Schwartz and Zanchetti demonstrated that coronary occlusion could cause an increase in preganglionic sympathetic activity to the heart (65). This reflex was present prior to and following spinal transection at the first cervical segment. In 1971, Brown and Malliani (19) showed that, in vagotomized cats with C1 spinal transection, increases in coronary arterial pressure could cause increases in activity of both preganglionic and postganglionic sympathetic nerves to the heart. In addition, Malliani et_a1, (60) showed that increases in systemic arterial pressure could alter preganglionic sympathetic activity in vagotomized, spinal cats. Previously, Coote_et.gl. (22) had shown that the size of sympathetic reflexes was reduced after decere- bration, but that reflexes were returned to initial or greater size upon high Spinal transection. In considera- tion of Coote's findings, which indicated that supraspinal centers could have inhibitory influences on sympathetic reflexes, Brown and Malliani (l9) postulated that a spinal sympathetic reflex arc might "represent the most elemen- tary organization for circulatory reflexes, upon which supraspinal influences exert their effects." The concept of spinal sympathetic control of circu- lation was not a new one. Fernandez de Molina and Perl (30) had noticed periodic changes in sympathetic pregan- glionic discharge and systemic arterial pressure in spinal cats which led them to suggest sympathetic control of the circulation at the spinal level. The first report of a spinal sympathetic reflex is credited to Sherrington. In his Mammalian Physiology, he stated that stimulation of the splanchnic nerve in the spinal cat caused increases in arterial pressure (94). In 1924, Langley (53) confirmed Sherrington's observation. He also showed that increases in pressure could be evoked by stimulation of somatic nerves, indicating the existence of a spinal somato-sympathetic reflex arc. However, Lang- ley pointed out that the increases evoked by somatic stimu- lation were much smaller and concluded that the splanchnic nerve likely contained more afferent fibers capable of evoking vascular reflexes. More evidence of spinal sympatho-sympathetic reflexes was presented by Downman and McSwiney (28). By pinching the abdominal viscera in spinal cats, these workers evoked large increases in arterial pressure. Mukherjee (74) then demonstrated that bladder distension in spinal cats could reflexly increase arterial pressure. In this study, it was pointed out that bladder distension also could cause renal vasoconstriction. Up to this point, the ability of the sympathetic efferent system to respond to peripheral stimulation in the spinal animal had been shown only indirectly. Arterial pressure changes could be induced, but sympathetic efferent nerve activity had not been recorded. In 1964, Beacham and Perl provided direct evidence that the sympathetic efferent system was capable of responding reflexly in spinal animals (5). These investigators showed that afferent stimulation of dorsal roots, spinal nerves or limb nerves was capable of inducing a reflex discharge in sympathetic preganglionic fibers exhibiting tonic activity. However, possibly the most interesting finding was that both excit- atory and inhibitory responses could be evoked. The obser- vations that some of these neurons are tonically active and are capable of responding with an increased or decreased discharge greatly increase the possibilities for spinal I sympathetic control of circulation. The recent information provided by investigators in Malliani's laboratory makes the hypothesis of spinal sym— pathetic control of circulation extremely attractive. The work that most immediately prompted the hypothesis was the 10 demonstration of sympathetic discharge upon coronary occlu- sion (65) or increases in coronary or systemic arterial pressure (19,60) in spinal animals. Subsequently, Peter- son and Brown (85) showed that, in spinal cats, central stimulation of either the inferior cardiac or pericoronary nerves could increase arterial pressure without effect on heart rate. Following the observation by Peterson and Brown, Malliani_gt a1, (62) were able to show that dP/dt max could be reflexly increased in spinal animals by stimulation of the afferent fibers in cardiac sympathetic nerves. dP/dt max refers to the maximum rate of change in ventricular pressure with time and is considered a fairly accurate in vivo measurement of myocardial contractility (14). As the authors pointed out, increases in dP/dt max can result from changes in heart rate, changes in preload (ventricular pressure at the end of diastole), or changes in afterload (aortic pressure) (62). In this set of experi- ments neither heart rate nor preload changed. The authors felt that only a negligible contribution to the increase in contractility was made by changing afterload, since afferent stimulation produced no change in arterial systolic pressure. Also, increases in arterial systolic pressure were detect- able only when dP/dt max had already reached peak value. It was concluded that the increase in myocardial contrac- tility was reflex in nature because there were no direct connections between stimulated afferent fibers and efferent ll fibers involved in the pathway, and also because a direct effect such as alteration in heart rate, preload or after- load, could be excluded. A further study by Malliani 33 31. (61) showed that either chemical activation of cardiac sympathetic receptors, or electrical activation of cardiac sympathetic afferent nerves could cause a tachycardia. The tachycardia was reflexly evoked in cats with neuraxis intact and in cats with neuraxis severed at C1. Stimulation also caused a transitory apnea and an increase in arterial pressure. Since removal of the right stellate ganglion eliminated the tachycardia without affecting the increase in arterial pressure or change in respiratory pattern, it was proposed that the right stellate ganglion was required for the effer— ent pathway. Also, since interruption of the upper 4 thoracic rami communicantes (through which afferent sym- pathetic fibers gain entry into the spinal cord through the dorsal roots) eliminated all responses to cardiac sym~ pathetic afferent stimulation, it was suggested that these nerves form the afferent limb of the reflex. Finally, in 1974, workers in Malliani's laboratory showed that, in spinal, vagotomized cats, sympathetic preganglionic fibers from the third or fourth thoracic sympathetic ramus could respond reflexly with either increases or decreases in frequency of discharge depending on the location of stimulated receptors (83). Specifi- cally, activation mainly of cardiac receptors always 12 reflexly increased discharge of responsive preganglionic fibers. In contrast, simultaneous activation of cardiac and vascular receptors or activation of aortic receptors could either increase or decrease discharge of pregan- glionic fibers, although each fiber was consistent in response. Thus, much evidence has accrued in support of the hypothesis that the sympathetic nervous system is at least capable of reflexly altering cardiovascular function without input from higher centers. Possibly, the recently described cardiopulmonary- renal sympathetic reflex (101) also contains a component which is mediated at the spinal level. Changes in renal nerve activity could be effected rapidly by alterations in cardiopulmonary receptor activity and would not necessar- ily require input from the brainstem. Effect of Renal Nerves on Renal Sodium and Water Excretion: Renal nerves may contribute significantly to the control of renal function. Since Bernard first reported a diuresis after sectioning the splanchnic nerve in an anesthetized animal (12), the effect of renal nerve activity on the kidney has been in question. One of the first attempts at complete renal dener- vation was made by Marshall and Kolls (68,69) who sectioned the splanchnic and all visible renal nerves. They found that, in dogs, either unilateral adrenalectomy, unilateral splanchnicotomy or unilateral section of all visible renal nerves resulted in a dilute urine in the denervated kidney, 13 such that the total amount of water, chloride, and urea excreted by the denervated kidney was greater than that excreted by the intact kidney. However, there was little difference in total creatinine or phenolsulphonphthalein excreted. Many of these effects were eliminated by applying pressure to the artery of the denervated kidney with a pressure cuff. However constricting the renal artery in this manner, also resulted in decreased total creatinine excretion. The authors attributed the changes in excretion of urea and sodium chloride to alterations in blood flow. However, neither creatinine excretion, (a measure of GFR), nor phenolsulphonphthalein (an indicator of renal blood flow) were increased as a result of denervation. Due to the failure of Marshall and Kolls to demon- strate an increased GFR or renal blood flow in response to renal denervation, Kriss et a1. (51) repeated the study using mannitol as a measure of CPR and para—amino hippurate (PAH) as a measure of renal plasma flow. These workers showed that unilateral splanchnicotomy resulted in a 447% increase in chloride excretion and a 205% increase in water excretion from the denervated kidney compared to the control kidney. In contrast, mannitol and PAH excretion were increased only 18% and 19% respectively, in the denervated kidney. These authors stated that, although the increases in chloride and water excretion could not always be explained by increases in glomerular 14 filtration rate or renal plasma flow, there was not enough evidence to conclude that alterations in excretion were due to specific inhibition of tubular reabsorption. In 1951, Kaplan and Rapoport (47) again examined the effects of splanchnicotomy on renal function under conditions of osmotic diuresis. These investigators used creatinine as an index of CPR and PAH as an index of renal plasma flow. Creatinine is generally believed to be a better indicator of GFR than is mannitol (11). They showed that sodium and chloride excretion were in- creased in the denervated kidney although neither GFR nor renal plasma flow were increased above values determined in the innervated kidney. This indicated that changes did not result from alterations in filtered load. These authors pr0posed that the splanchnic nerve might control proximal tubular reabsorption of sodium. The major problem with acceptance of the hypothesis that renal nerves can alter renal tubular functions stems from the concept that even undetectable changes in GFR might alter sodium excretion. This argument was used by Selkurt (93) in 1954 concerning the work of Kaplan and Rapoport. Selkurt pointed out that the GFRs in Kaplan’s and RapOport's dogs were always higher in the denervated kidney. In fact, this was the case although in one group of experiments, GFR was increased only 3% in the dener- vated kidney, whereas sodium excretion in the denervated kidney was increased 102% over that in the innervated lS kidney. Thus, in spite of accumulating evidence, most investigators refused to accept the hypothesis that renal nerves could influence renal tubular reabsorption. Another criticism was that differences in renal func- tion following renal denervation could only be demonstrated in anesthetized dogs. Studies in unanesthetized dogs had failed to show differences between innervated and denervated kidneys (42,66,89). In 1952, Berne (l3) conducted a study comparing the effects of chronic unilateral denervation in anesthetized and unanesthetized dogs. He confirmed the findings of others that renal function does not differ between denervated and innervated kidneys in the unanes- thetized animal and felt that the differences he saw in anesthetized animals were due to differences in GFR. Kamm and Levinski (46) approached the problem by clamping the renal artery of a denervated kidney. The purpose of the clamp was to return sodium excretion to predenervation levels. When this was done, they found that the amount of sodium being filtered by the kidney also returned to predenervation levels. They reasoned that the denervation natriuresis must have resulted from an increase in filtered sodium, since clamping the renal artery caused filtered sodium to return to predenervation levels. Had decreased sodium reabsorption been the cause of denervation natri- uresis, clamping the renal artery should have caused filtered sodium to fall below the predenervation level. More 16 credence was added to the idea that renal nerves did not affect renal tubular reabsorption when using histofluor— escence techniques, Nilsson failed to demonstrate adren- ergic innervation of the renal tubules in rats and rabbits (77). This finding was subsequently confirmed in dogs (71). The Kamm and Levinski study seemed to be rather con- clusive. However, as pointed out later by Bonjour et a1. (15), Kamm and Levinski failed to report urine volume. Possibly, renal denervation produced a diuresis in their experiments. Also, a fall in renal arterial blood pressure might in itself be antinatriuretic independent of changes in GFR. By microsphere injection, Bonjour et a1. were able to decrease GFR in a denervated kidney without alter— ing renal arterial pressure. Using this procedure they found that even when GFR was decreased 40% there was still a significant diuresis and natriuresis in the denervated, as compared to the innervated kidney. Apparently, alter- ations in GFR could not necessarily account for changes in function following denervation. Renal denervation.results not only in alterations in GFR but also in alterations in renin concentrations. In 1964, Taquini et al. ( 99) showed large differences in renin concentrations in innervated versus chronically denervated rat kidneys. At the same time, Barajas (3) showed that the juxtaglomerular apparatus was innervated. Possibly the increased sodium excretion seen following 17 denervation was due to decreased renin secretion, since a decrease in circulating renin would result in a decrease in angiotensin 11 production, and this, in turn would lead to a decrease in release of aldosterone. This, of course, would indicate an indirect effect of renal nerves on renal sodium excretion through the renin-angiotensin system. Another possible mechanism through which renal nerves might alter sodium and water excretion, concerns blood flow distribution. Aukland (1) investigated the effects of renal nerve stimulation on blood flow distribution and found that, during renal nerve stimulation, both cortical and medul- lary flow were reduced to the same extent as measured by local clearance of hydrogen gas. Using a technique 85Kr autoradiographs, workers in another labora- involving tory found that stimulation of renal nerves resulted in relative increases in outer medullary blood flow (86). Bencsath and Takacs (10) approached the problem by examin- ing the effects of unilateral splanchnicotomy on intra« renal distribution of blood flow in hydropenic, normo- volemic, isotonic volume expanded and hypotonic volume expanded dogs. They found, using isotope indicators, that denervation resulted in an increased medullary blood flow in the denervated kidney especially in the hydro- penic and normovolemic animals. However, although the increase in medullary flow was less in animals whose blood 18 volume had been increased, these animals showed a much higher level of diuresis and natriuresis than the hydro- penic and normovolemic animals. They concluded that, although the diuresis seen following denervation might be due to increases in medullary blood flow, the natri- uresis was not due to alterations in blood flow distri- bution. Again, it was suggested that renal nerves might exert a direct influence on renal tubules, or an indirect influence other than that associated with changes in blood flow. This hypothesis was supported by work previously done by Gill and Bartter who showed that adrenergic block- ade failed to decrease sodium excretion in patients on low sodium intake, although GFR was decreased and plasma aldosterone concentration was increased (33). Similarly, Gill and Casper showed that stimulation of alpha adrenergic receptors in the kidney resulted in increased reabsorption of sodium (34). Alpha adrenergic stimulation was accom- plished by infusing the kidney with norepinephrine and propranolol (a beta adrenergic blocker). Gill and Casper suggested that the decrease in urine flow and free water clearance along with the lack of change in GFR and urine osmolarity provided indirect evidence that alpha adrenergic stimulation increased proximal tubular sodium reabsorption. Bello-Reuss 3£_31, (7) then showed by micrOpuncture that the increase in sodium excretion in denervated rat kidneys was due to decreased reabsorption 19 in the proximal tubule which was only partially compen- satedfor by more distal segments of the nephron. In this study, GFR was unchanged. Prior to the study by Bello- Reuss 31_31,, Mfiller and Barajas (75) had shown with electron microscopy that both proximal and distal tubules were directly innervated. When one considers that renal alpha-adrenergic stimulation may increase proximal tubular sodium reabsorption, that renal denervation does decrease proximal tubular sodium reabsorption, that renal tubules are innervated, and also that norepinephrine can stimulate active transport of sodium in epithelia similar to that of renal tubules (4,39), the possibility of a direct action of renal nerves on renal tubules influencing sodium move- ment becomes quite plausible. Recently, it has been shown that direct low frequency (1-2 Hz) electrical activation of renal nerves in dogs can cause sodium retention without causing changes in GFR, renal blood flow or intrarenal blood flow distribution (95). Low frequency electrical activation of renal nerves in rats also was shown to cause an antidiuresis and antinatriuresis without changes in GFR or renal plasma flow (9). The study in rats which used whole kidney and single nephron tech- niques showed that the response appeared to be mediated by slowly conducting (0.7-1 m/sec), unmyelinated efferent renal nerves and that effects seen were due to action of these nerves on the proximal tubule. These studies were 20 important since questions have arisen as to adverse effects of renal denervation. Katz and Shear (49) have shown assym- etric intra—renal blood flow distribution and renal damage resulting from denervation. They also suggested that dener- vation methods such as splanchnicotomy might result in incomplete denervation. Because renal nerve stimulation can cause the release of renin (52) and prostaglandins (29) from the kidney, possibly the decreases in sodium excretion are not due to direct catecholaminergic influences on the renal tubule. Since both angiotensin II (70) and prostaglandin E1 (38,56) increase sodium transport by toad bladder and frog skin, as does norepinephrine, (4,39) an indirect mechanism involving renal nerves still cannot be ruled out. Zambraski and DiBona (102) showed that the antinatri- uresis seen on low level renal nerve stimulation was not affected by angiotensin II inhibition. They suggested that the antinatriuresis seen with renal nerve stimulation was due to an increase in proximal tubular sodium reabsorption and was the direct result of the action of renal nerves on the proximal tubule. Kaloyanides 33 31. (45) then showed that the antinatriuresis could not be due to increases in prostaglandin by pretreating with indomethacin, a prosta- glandin synthetase inhibitor. Thus, attention was turned back to direct catecholaminergic influences on the renal tubule. 21 Previously, Gill and Casper had shown that stimulation of alpha adrenergic receptors in the kidney caused decreases in sodium and water excretion (34). Recently, workers in DiBona'a laboratory have reexamined the specificity of the adrenergic effect with micropuncture techniques, using low frequency renal nerve stimulation in dogs (103). Their results showed that the antinatriuretic response to renal nerve stimulation could be blocked by phenoxybenzamine, an alpha adrenergic receptor blocking agent. A subsequent study by the same laboratory demonstrated that antinatri- uresis evoked by reflex stimulation of the renal nerves also could be blocked by phenoxybenzamine or by guan- ethadine(104). Since guanethadine does not inhibit the response to circulating norepinephrine but specifically affects responses to sympathetic adrenergic stimulation, and phenoxybenzamine specifically blocks alpha-adrenergic receptors (37), the indication is that sodium reabsorption may in part be controlled by alpha adrenergic activity of renal nerves with endings directly on the tubules. Recent denervation studies in rats (8) and dogs (82) support the hypothesis that renal nerves may directly alter sodium excretion by acting on the proximal tubule. RATIONALE In cats with an intact neuraxis, electrical stimulation of cardiopulmonary sympathetic afferent nerves causes an excitation of renal sympathetic efferent nerve activity folloWed by an inhibition (101). Activation of these afferent nerves also has been shown to alter cardiac sym- pathetic efferent nerve activity via spinal and supraspinal pathways. Therefore, experiments were designed to determine the existence of a purely spinal component of the cardio- pulmonary-reflex. Also, since activation of cardiopulmonary sympathetic nerves can alter renal nerve activity (101), and alterations in renal nerve activity may alter renal function (27), experiments were designed to determine if cardiOpulmonary sympathetic afferent stimulation was capable of altering renal function. 22 METHODS GENERAL METHODS Experiments were conducted in 33 cats. One cat was anesthetized with sodium pentobarbital (60 mg/kg) to allow spinal cord transection but was unanesthetized during the experiment. Thirty-two cats were anesthetized either with alpha chloralose (60 mg/kg) or with a mixture of sodium diallyl-barbiturate (50 mg/kg), urethane (200 mg/kg), and monoethyl urea (200 mg/kg). Cats of either sex were used and body weight ranged between 2-3 kg. A tracheostomy was performed in all cats and both fem—. oral veins were cannulated for delivery of drugs and infu- sates. One femoral artery was cannulated for monitoring arterial pressure and a second femoral artery was cannulated for withdrawing arterial blood samples. In some experi- ments the left jugular vein was cannulated with cannula advanced close to the right atrium for monitoring central venous pressure. Pressures were monitored with a pressure transducer (Model P23A; Statham, Inc. Hato Rey, P.R.) connected to a Grass polygraph (Model 7; Grass Inst. Co, Quincy, MA). To insure adequate muscle relaxation , cats were immobilized with 4 mg/kg gallamine triethiodide (Flaxedil, Davis-Geck, Pearl River, NY) and artificially respired with a Harvard respirator (model 607; Harvard Apparatus Co., Millis, MA). Subsequent doses of Flaxedil were administered as needed, following assessment of level of anesthesia. Respiration frequency was 18-20 breaths_per 23 24 min. Tidal volume was approximately 45 ml and was ad- justed to insure normal arterial P02 and pH as measured with a blood gas analyzer (Radiometer, Model MKZ; COpen- hagen). Arterial PCOZ often was lower than normal due to slight hyperventilation. ESOphageal temperature was moni- tored with a Telethermometer (model 43TD; Yellow Springs Inst. Co., Yellow Springs, OH) and maintained between 360 and 38°C with lamps and heating pads. Influences of carotid sinus, aortic arch, and vagally innervated baro- and chemoreceptors were eliminated by severing glossopharyngeal and vagus nerves bilaterally at the jugular foramen. In some of the spinally transected cats, only the vagus nerve was severed, since severing the neuraxis eliminated reflex influences of the glOSSOpharyngeal nerve on renal nerve activity. In all cats access to the kidney was gained through a retroperitoneal approach. In most cats, one or two multi- fiber branches of a left renal nerve were severed and the central end was tied with saline—soaked thread. Nerves were tied with thread to facilitate attachment to electrodes. Nerves were immersed in a pool of mineral oil to prevent drying. In one group of experiments the left ureter was cannulated for collection of urine samples. The left stellate ganglion was isolated in all experi— ments which utilized electrical stimulation of afferent cardiac sympathetic nerves. The ganglion was approached retropleurally after removal of portions of the second 25 and third ribs. Leaving the pleura intact, cardiac nerves emanating from the caudal edge of the ganglion (inferior cardiac nerve and/or ansa subclavia) were severed and the central ends were tied with saline-soaked thread. This procedure facilitated attachment of nerves to electrodes. Nerves were subsequently immersed in a pool of mineral oil SPECIFIC METHODS I: SPINAL REFLEX EXPERIMENTS (FIGURE 1) A. Activation of Afferent Nerves 1. Activation by Electrical Stimulation In 7 cats cardiopulmonary or renal sympathetic affer- ent nerves were activated by electrical stimulation. The severed, tied central end of a cardiac or renal nerve was laid across one pole of a bipolar platinum electrode, with the attached thread loop being placed over the second pole. This technique allowed good contact of the nerve with the electrode and prevented the nerve from slipping. Nerves were stimulated with trains of pulses. A train was 10 msec in duration and consisted of 3 equally spaced, square wave pulses. Each pulse was 0.5-1.0 msec in duration and 8—30V in intensity. Train frequency was 0.5 per sec. Stimuli were delivered to the affer- ent nerve from a Grass stimulator (Model S48) through a capacitance-coupled Grass stimulus isolation unit (Model SIUS). 2. Activation by Intravascular Volume Expansion In 7 cats, cardiopulmonary sympathetic afferent 26 nerves were activated by increasing the circulating blood volume. All cats received an infusion of 3% dex- tran in isotonic saline. The infusion was delivered into a femoral vein at 4.4 ml per min using a Harvard infusion pump (Model 975; Harvard Apparatus Co.). The blood volume of 4 cats was expanded with 25 ml dextran per kg; the blood volume of 3 cats was expanded with 15 ml dextran per kg. Neural Recording_ The severed, tied central end of a cardiac or renal nerve was laid across one pole of a bipolar platinum electrode, with the attached thread loop being placed over the second pole. Spontaneous or evoked activity of efferent cardaic or renal nerves detected at the recording electrode was amplified by a capacitance- coupled Grass preamplifier (Model P511) using a band— width of 30 or 100 Hz - 1K Hz, displayed on a Tech- tronix oscilloscope (model D13; Techtronix Inc., Beaver- ton, OR), and stored on magnetic tape with a Tandberg recorder (series 100; Sangamo Data Systems, Columbus, OH, supplier). Protocol 'Responses to Activation of Afferent Nerves with Elec- trical Stimulation Efferent nerve responses to cardiopulmonary sym- pathetic afferent stimulation were compared prior to 27 and following C1 spinal cord transection. Prior to spinal cord transection, the ansa subclavia or inferior cardiac nerve was electrically stimulated and renal nerve activity was recorded in S anesthetized cats to verify the presence of a cardiopulmonary—renal sympathetic reflex. In two of these cats the presence of a cardio-cardiac reflex was verified by stimulating the ansa subclavia while recording from the inferior cardiac nerve. Similarly, for determination of a renal- renal reflex, one branch of a renal nerve was stimulated while evoked activity was recorded from a second branch of a renal nerve. Frequency of stimulation in all cases was 0.3-0.5 trains per sec and intensity was 8-30V. To insure that stimulus voltage was supramaximal, voltage was increased until the amplitude of the evoked efferent nerve response could not be increased further by further increases in stimulus voltage.‘ Following initial characterization of reflexes, the spinal cord was exposed at the first cervical seg- ment (C1). Immediately prior to spinal transection the dura was Opened, taking care not to interrupt any venous sinuses. The spinal cord was then severed at 'C1 using blunt dissection. The spinal cord also was severed in two cats in which reflexes were not char- acterized prior to spinal transection. Warmed dextran (3% in isotonic saline) or phenylephrine (0.06% Neo- 28 Synephrine, Winthrop Laboratories, NY, NY) were infused into a femoral vein to maintain arterial pressure above 70 mmHg, if necessary. Infusion rates were 2 ml/min and 0.01 ml/min, respectively. Transection of the spinal cord often causes a marked but transient depression of spontaneous nerve activity. Following the recovery of spontaneous nerve activity, attempts were made to evoke a cardiOpulmonary- renal reflex. In two cats, the presence of'a cardio- cardiac reflex, and in three cats the presence of a renal-renal reflex also were determined. At the end of the experiment hexamethonium. (a ganglionic block- ing agent) was given to block neural transmission and thus allow accurate assessment of noise. Responses to Activation of Afferent Nerves with Intra- Vascular Volume Expansion In 7 cats, in which the spinal cord was transected, cardiopulmonary sympathetic afferent nerves were acti- vated by intravascular volume expansion in a further attempt to elicit a spinal cardiopulmonary-renal re- flex. The spinal cord was transected at C1 using blunt dissection. Arterial pressure was monitored and in cats 'whose blood pressure was less than 70 mmHg, pressure was supported with a phenylephrine infusion (0.06% Neo- Synephrine) at 0.01 ml/min. As previously described, nerve activity is often markedly depressed following 29 spinal cord transection. Thus, time was allowed for activity to recover. Following recovery of spontan- eous nerve activity intravascular volume expansion (as described in Specific Methods LA.2) was begun. During the infusion, renal nerve activity was sampled every 2 min. Following the end of infusion, activity was sampled every 2 min for 15 min and every 5 min thereafter, for at least one hour. Throughout the experiment central venous pressure was contin- uously monitored. At the end of the experiment hexa- methonium was given to block neural transmission and thus allow accurate assessment of noise level. Data Analysis 1. Responses to Activation of Afferent Nerves with Electrical Stimulation Nerve activity stored on magnetic tape, was later characterized by constructing histograms with the use of a Nicolet computer (Model 1070; Nicolet Inst. Corp., Madison, WI). Prior to computer analysis, nerve acti— vity was processed by a window discriminator. Absolute noise level (10-20 uV) which was recorded at the end of each experiment, was used to facilitate prOper adjust- ment of the threshold of the window discriminator. The threshold was considered to be adjusted properly when the window discriminator generated a pulse each time the voltage due to nerve activity exceeded that due to noise. These pulses were counted by the computer. 30 During electrical stimulation, a trigger signal from the stimulator was recorded simultaneously with efferent nerve activity. This signal, which corres- ponded in time with each stimulation of the afferent nerve, was used to trigger the computer to count pulses generated by the window discriminator. An internal trigger was used to initiate computer count- ing of spontaneous nerve activity. Once triggered, the computer was set to count for 200 msec or 1 sec. Histograms constructed consisted of summations of 50 or 100 triggered responses (evoked or spontaneous nerve activity). Histograms were then plotted with an x-y recorder (Model 7015A; Hewlett-Packard, San Diego, CA). Responses to Activation of Afferent Nerves with Intra- vascular Volume Exp3nsion Nerve activity stored on magnetic tape was later processed by computer to determine frequency of dis- charge. Basic computer techniques are described in Specific Methods I.D.L In these experiments the com- puter was used to sample and count spontaneous nerve activity for 30 sec periods. The number of counts registered by the computer was then displayed as a digital readout and used to calculate discharge fre- quency in counts per sec. 31 Mean control discharge frequency was calculated from four to six 30 sec samples obtained over a 5 min period. Following the initiation of intravascular volume expansion, discharge frequency was calculated every 2 min. Discharge frequencies obtained during intravascular volume expansion were transformed into percent of control discharge frequency. Central ven- ous pressure also was recorded during the experiment. Following initiation of intravascular volume expansion, change in central venous pressure was determined at two minute intervals simultaneously with estimates of nerve activity. Data were analyzed by constructing a confidence interval (x f t - Si) around central venous pressure (changes from control) or renal nerve activity (percent of control). Responses not contained within the confi- dence limits were considered significantly different from control (P‘< 0.05). SPECIFIC METHODS II: RENAL FUNCTION EXPERIMENTS (FIGURE 2) A. Activation of Afferent Nerves In three groups of cats, cardiopulmonary sym— pathetic afferent nerves were prepared for electri— cal stimulation as described in Specific Methods I.A.l. In one group of cats, the afferent nerve was prepared for stimulation but was not stimulated. This group constituted a control group. 32 In a second group of cats afferent nerves were stimulated at 1-2 trains per sec. (Trains have been previously described in Specific Methods 1. A.l.) Stimulus intensity in these experiments was 8—12V. In a third group of cats, afferent nerves were stimulated at 5-7 Hz. Stimulus duration was 0.5-1 msec and in- tensity was 8-12V. Neural Recordigg The effect of afferent nerve stimulation on renal efferent nerve activity was assessed in 7 cats. Nerves were prepared as previously described (Specific Methods I.B) and activity was amplified by a capacitance-coupled Grass preamplifier (Model 7) using a bandwidth of lOHz- 500Hz. Activity was displayed on a Grass polygraph (Model 7). Sample Collection for Estimation of Renal Function Urine and blood samples were obtained to allow estimation of glomerular filtration rate (GFR), urine flow rate, sodium excretion, and potassium excretion. Inulin clearance was measured to assess GFR. A solution of inulin (3% in isotonic saline) was infused into a femoral vein at 0.299-0.419 ml/min. Infusion 'was begun one hour prior to the initiation of an experi- ment to insure a constant plasma inulin concentration. Urine samples were collected from a cannula inserted into the left ureter. Urine collection periods were 33 10 min. Blood (1.5 ml) was sampled at the midpoint of each collection period from a cannula inserted into a femoral artery. Plasma was then separated from the blood sample and stored for later analysis. Protocol Experiments were conducted in three groups of cats to assess the effect of cardiopulmonary sympa- thetic afferent stimulation on renal function. In a control group of 6 cats, consecutive urine and blood samples were obtained for 40-70 min. In cats in which afferent nerves were stimulated (1-2 trains/sec. or 5-7 Hz), after collecting two or .three consecutive urine samples of constant volume, afferent stimulation was begun. Urine and blood samples were collected during the 10 min stimulation period and during two or three post-stimulation periods. Arter- ial pressure was continuously monitored in all cats and stabilized, if necessary, by infusing or withdrawing of a small volume (2 ml) of blood. Renal efferent nerve activity was continuously recorded in S of 7 cats in which afferent nerves were Stimulated at l-2 trains/sec. Renal nerve activity also was recorded from '2 additional cats in which afferent nerves were stimu- lated at 5-7 Hz. Renal function parameters were not measured in these 2 cats. 34 E. Data Analysis l. Neural Recordingg Renal nerve activity was displayed on a Grass poly- graph as described in Specific Methods II.B. This acti- vity was simultaneously quantified by cumulative inte- gration with a Grass integrator (Model 7P10). The threshold of this integrator was adjusted until only that voltage which exceeded noise level was accumulated. Voltage was integrated until a given preset voltage was reached. At this time the integrator reset and began accumulating voltage again. Changes in nerve activity were reflected as changes in time required for inte- grator resetting. This time is referred to as epoch time. Thus, inhibition of nerve activity would lengthen epoch time. Conversely, excitation of nerve activity would shorten epoch time. 2. Renal Function Urine and plasma samples were analyzed for Na+ and K+ concentration by flame photometry with a Beckman photometer (Model 105; Beckman Inst., Palo Alto, CA). Urine and plasma inulin concentrations were determined by the method of Schreiner (92) (see Appendix). Total ‘excretion of Na+ (UNaV) and K+ (UKV) were calculated as the product of ion concentration in a given urine 35 sample and the urine flow rate. Fractional excretion of Na+ (FENa), which is the amount of Na+ excreted per amount filtered, was calculated as: FE = UNa - V GFR ° Na 100 PNa - where UNa urine sodium concentration (qu/ml) "U I Na - plasma sodium concentration (qu/ml) V = urine flow rate (ml/min) GFR glomerular filtration rate (ml/min) GFR was calculated as Uin ' V Pin where Ui urine inulin concentration (mg%) n Pin = plasma inulin concentration (mg%) V = urine flow rate (ml/min) Data from the cats undergoing afferent stimu- lation were statistically analyzed using a randomized complete block design (96). Grouped data were expressed as a mean and variation was estimated with a coeffi- cient of variability. Means were compared using the least significant difference test. The control state for each cat was estimated by averaging data obtained during the 10 min collection periods prior to stimula— tion. Similarily the data obtained during collection 36 periods following stimulation were averaged. 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D 85...... 2.883.882 o 2 8331.832 o 28o... 3.889 c 1‘ H H 5......5.......... H H 750 11 1 H .. H H .58 1 H H H H .5 ..00 \ 67 0.N 0.0 - 0.02 592...: .32 00¢ 03. 03 00—. 09 0.N 0.? 0.0 0.0 68 Figure 13. Summary of renal responses to cardiopulmonary sympathetic afferent stimulation at 1—2 trains/sec in seven cats. Control data are indicated by black bars, responses to stimulation are indicated by stippled bars and post- stimulation data are indicated by striped bars. Abbre- viations are the same as those defined in Figures 11 and 12. Data are expressed.as a mean.Coefficient of variation also is given. The number of replications is seven. . Control for each cat consists of averaged data obtained from two or three samples collected prior to stimulation. The post-stimulation sample for each cat consists of averaged data obtained from two or three samples collected following stimulation. Stimulation data were obtained from the one sample collected during stimulation. Statistical signi- ficance (*) was attained at P < 0.05. Both UN V and FE were increased significantly during and after gtimulation. Urine flow rate was significantly increased following stimulation. In contrast, UKV’ GFR and MAP did not change. 69 UNaV (qu/min) UKV (qu/min) 20 * * :7 1O é o c s P c.v.=24.9°/. c.v.= 34.0% FENa (7.) Ci" (ml/min) 5-0 2.5 0 c.v.=17.3°/o V (uI/minf- BP (mmHg) 150 100 O c S P c.v.=23.8°/o 70 Figure 14. Summary of renal responses to cardiopulmonary sympathetic afferent stimulation at 5-7 Hz in 5 cats. Control data are represented by black bars, responses during stimulation are represented by stippled bars, and responses following stimulation are represented by striped bars. Abbreviations are the same as those described in Figures 11 and 12. Data are expressed as a mean. Coefficient of variability also is given. Control for each cat is averaged data obtained from two or three samples collected prior to stimulation. 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