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Kidney International, Vol. 44 (1993), pp. 684—691

Juxtamedullary afferent and efferent arterioles constrict to renal nerve stimulation

JING CHEN and JOHN T. FLEMING

Department of Physiology and Biophysics, School of Medicine, University of Louisville, and Center for Applied Microcirculatory Research, Louisville, Kentucky, USA

Juxtamedullary afferent and efferent arterioles constrict to renal nerve preferential preglomerular action may not be applicable to the stimulation. Sympathetic neural control of afferent and efferent arteri- arterioles of inner cortical (juxtamedullary) glomeruli. oles of inner cortical (juxtamedullary) glomeruli has not been estab- lished, in part, because of difficulty accessing these vessels, normally A reduction of medullary blood flow during renal nerve located deep below the surface. In this study we utilized the rat stimulation [15] confirms the histological evidence for innerva- hydronephrotic kidney model to visualize the renal microcirculation tion of vessels in the inner cortex [2, 3, 12, 14, 20]. However, and to quantitate the responses ofjuxtamedullary arterioles to brief (30 these data do not provide evidence for the relative response of see) renal nerve stimulation (RNS). Juxtamedullary afferent and effer- ent arterioles constricted in a frequency-dependent fashion to RNS, the pre- versus postglomerular vessels to nerve stimulation. achieving a maximal constriction of 35% to 8 Hz stimulation. In these According to Gorgas [14], the juxtamedullary efferent arterioles same kidneys, outer cortical also constricted to RNS are surrounded by a denser nerve plexus than cortical efferent but outer cortical efferent arterioles did not. Microinjection of norepi- arterioles. Barajas and Powers [3] have reported that the length nephrine (NE) around single outer cortical efferent arterioles (to avoid the constriction of preglomerular vessels) constricted the efferentof adrenergic innervation is considerably longer among jux- arterioles. However, the afferent arterioles of the same glomeruli were tamedullary efferent arterioles than efferent arterioles of super- considerably more responsive to microinjected NE. Thus, the lack of ficial . Thus, the adrenergic innervation of juxtamed- constriction of outer cortical efferent arterioles to RNS may relate, inullary efferent arterioles appears to be more extensive than part, to their low sensitivity to NE, the primary neurotransmitter. These direct observations indicate that the juxtamedullary efferentouter cortical efferent arterioles. Since the walls of juxtamed- arterioles are responsive to renal nerve stimulation whereas the outer ullary efferent arterioles are more muscular than outer cortical cortical efferent vessels are not. These results, which should beefferent arterioles [2, 10, 19, 25], the juxtamedullary vessels cautiously extrapolated to normal filtering kidneys, indicate that gb- may have a greater capacity to respond to nerve stimulation. merular hemodynamic changes evoked by the sympathetic nervous system are different for outer cortical and inner cortical glomeruli. Juxtamedullary efferent arterioles are known to respond to norepinephrine [6, 17, 281, the principal sympathetic neuro- transmitter. However, responses to exogenous norepinephrine may not correlate with the microvascular constrictor pattern to Though not essential for normal renal function, the sympa-neural stimulation. thetic nerves of the mammalian kidney play an important role in The primary objective of this study was to quantitate the mediating changes in renal function via glomerular hemody-constrictor responses of juxtamedullary afferent and efferent namic and tubular reabsorption changes under altered physio-arterioles to electrical stimulation of the renal nerve and to logical (environmental stress, salt loading) and pathologicalcompare the responses against their outer cortical counterparts. (hypertension) states [8]. Regarding the renal vascular effects,The rat hydronephrotic kidney preparation provides a unique increases in efferent renal nerve activity elevate preglomerularmodel to accomplish this objective because it is possible to vascular resistance of outer cortical glomeruli to a greaterdirectly visualize the arterioles of juxtamedullary and outer extent than postglomerular resistance [1, 18, 22—24, 31, 32].cortical glomeruli [28]. Bührle and colleagues [5] confirmed Similarly, in the rat hydronephrotic kidney model which per-histologically that innervation of mice hydronephrotic kidneys mits direct visualization of the arterioles of outer corticalwas intact, and we have demonstrated that stimulation of the glomeruli, the preglomerular arterioles, but not the efferentrenal nerve induces frequency-dependent reduction in blood arterioles, give frequency-dependent constriction to brief renalflow to rat hydronephrotic kidneys [13]. Thus, the microvascu- nerve stimulation [13]. Thus, within the outer cortical microcir-lar and neural components are intact and functional in this culation, increases in renal nerve activity appear to have amodel. Another advantage of this model is the ability to observe preferential action on preglomerular vessels, However, such aarteriolar responses to very brief periods of electrical stimula- tion (30 seconds) to avoid eliciting non-neural factors, such as II formation, which alone can constrict pre- and Received for publication January 8, 1993 postglomerular vessels [221. and in revised form May 7, 1993 Our study also addressed why the outer cortical efferent Accepted for publication May II, 1993 arterioles of hydronephrotic kidneys are not responsive to © 1993 by the International Society of Nephrology nerve stimulation. We considered that the weak constriction to

684 Chen and Fleming: Neural control of juxtainedullary arterioles 685 nerve stimulation may reflect their weak reactivity to norepi-based on the system magnification factor determined by dis- nephnne, the primary neurotransmitter released from the nerveplaying the image of a stage micrometer on the monitor screen. varicosities. In several studies [17, 21, 27, 28, 30, 321 in which the renal microvascular network remained intact, the outer Arterlolar responses to renal nerve stimulation cortical efferent arterioles were less reactive to norepinephrine Juxtamedullary glomeruli were distinguished from outer cor- than the afferent arterioles. However, isolated and individuallytical glomeruli based on the length of the efferent arterioles. The perfused cortical efferent and afferent arterioles are equallyglomerulus was initially selected based on having rigorous reactive to norepinephrine [10, 35]. Hence, we reasoned thatblood flow through all loops and well-defined afferent the constriction of cortical preglomerular vessels could evoke aand efferent arteriolar walls for precise diameter measurements. myogenic or metabolic dilator mechanism to offset the directAfter a control period for diameter measurements, the renal constrictor action of norepinephrine on the efferent arterioles.nerve branch was stimulated at 8 Hz for no more than 30 To avoid upstream , norepinephrine was mi-seconds to determine if the afferent arteriole of the selected croiontophoretically injected around single outer cortical effer- constricted by at least 20%. Afferent arteiolar ent arterioles. The response of the afferent arterioles to micro-constriction confirmed that the nerves around the afferent injected norepinephrine was also quantitated for comparisonarteriole were activated by stimulating the single renal nerve with the efferent vessel responses. branch. If the afferent arteriole did not constrict, then another glomerulus was chosen. (This same criterion was applied to the Methods selection of outer cortical glomeruli.) After selecting a glomer- Induction of hydronephrosis ulus with a responding afferent arteriole, the nerve was stimu- lated at 2, 4 and 8 Hz (1.5 mA [15 VI, 1 ms, 30 sec) while Hydronephrosis was induced surgically by permanant liga- tion of the left among female Wistar rats (125 to 150 g)measuring the diameter of the afferent arteriole (within 100 zm anesthetized with Ketamine plus acepromazine. Six to eightfrom glomerulus) or the (within 100 sm from weeks later, the acute microvascular experiments were per-the glomerulus). Efferent arterioles were visualized as close to formed. the glomerulus as possible, but the site within 100 jzm of the glomerulus with the greatest optical resolution of the vessel Surgical preparation walls was selected to ensure the most accurate diameter mea- surements. Next, the stimulation was repeated while measuring On the day of the microvascular experiment, each rat wasthe diameter of the efferent arteriole at a site 500 and 1000 sm anesthetized with thiobutobarbital (mactin, 90 mg/kg, i.p.) anddownstream from the glomerulus. Then, an outer cortical placed on a heating pad to maintain rectal temperature betweenglomerulus with a responding afferent arteriole was selected 37 to 38°C. The trachea was cannulated to facilitate spontane-and the same procedure was repeated as described for the ous breathing. The right carotid artery and left jugular vein were juxtamedullary vessels. At each stimulation frequency. (2, 4 and catheterized for recording and administration of8 Hz) the diameter of the outer cortical afferent and efferent saline to supplement body fluid lost throughout the experiment.arteriole was measured within 100 sm from glomerulus. In all A pair of Teflon-coated stainless steel stimulating electrodesexperiments several minutes were allowed between nerve stim- was placed around the renal nerve branch which emerged fromulation periods. The maximum constriction during each stimu- the adrenal nerve, one of the three major renal nerves inner-lation period was measured by slow-motion playback of the vating rat kidneys [26]. The renal nerve was cut proximal to the electrodes to eliminate afferent conduction of electrical im-video tape. pulses. The other ends of the electrodes were connected to aOuter cortical and juxtatnedullary arteriolar reactivity to bath Grass stimulator (model S44) with a stimulus isolation unit for constant current output. The artery, vein and nerves to and norepinephrine from the left adrenal gland were ligated to exclude the possibil- Approximately 30 minutes after completing the nerve stimu- ity of catecholamine release from the adrenal gland during renallation protocol, norepinephrine was added to the bath to nerve stimulation. establish a 1 sMconcentration.Three minutes later, more norepinephrine was added to increase the bath concentration to The hydronephrotic kidney preparation 3 /LM.Theminimum arteriolar diameters were measured during The hydronephrotic kidney was split with a cautery andthe three-minute exposure to each bath norepinephrine concen- one-half of the kidney was exteriorized and outstretched in atration. Then the bath was drained and the kidney was washed bath. The interior surface of the split kidney was observed byseveral times with norepinephrine-free Kreb's solution and allowed the re-equilibrate for 30 minutes. light microscopy because juxtamedullary glomeruli are more prevalent on the internal side [28, 29]. The bath was sealed and Outer cortical arteriolar reactivity to micro-injected filled with a modified Kreb's solution warmed to 37 to 38°C and pH adjusted to 7.35 to 7.45 by bubbling carbon dioxide through norepinephrine the solution, The animal and intact kidney preparation were A glass micropipette (tip diameter < 2 jsm) filled with 0.1 mM placed on the stage of a light microscope to visualize thenorepinephrine was placed beside the outer cortical efferent or microvessels on the screen of a TV monitor by closed-circuitafferent arteriole. The norepinephrine was driven out of the television microscopy. The vessel images were recorded onpipette for no longer than 30 seconds by electrical pulses (1 Hz, video tape for measurement of diameter changes at a later time.900 ms, 0.2 mA) generated by the Grass stimulator, as previ- The actual in situ arteriolar lumen diameters were calculatedously described [9]. This procedure was repeated using a 686 Chen and Fleming:Neural control ofjuxtamedullary arterioles

Table 1. Outer cortical and juxtamedullary arteriolar dimensions and constriction to renal nerve stimulation and norepinephrine

NE-microiontophoresis Diameter Nerye stimulation % NE-bath % % jm Length jm 2 Hz 4 Hz 8 Hz I 3 M 0.1 mM 1.0mM

JM-AFF 18.6 1.4 93 3 79 365 5 99 1 77 3 JM-EFF 22.4 1.9 2,225 221 89 3 75 365 6 99 1 80 3 C-AFF 12.6 10b 93 2 78 465 5 86 4b 68 ioa 58 17 0 oa C-EFF 12.6 0.7" 254 20" 100 0 100 0 99 1b 93 3 85 7 87 5 48 8 Baseline arteriolar diameters (diameter) and total visible length (length) of juxtamedullary afferent (JM-AFF; N =11),juxtamedullary efferent (JM-EFF; N =II),outer cortical afferent (C-AFF; N =7)and outer cortical efferent (C-EFF; N =8)arterioles are given. Changes in arteriolar diameter induced by renal nerve stimulation (N =6),norepinephrine applied to the bath (NE-bath; N =6),and microiontophoretic application of norepinephrine (NE-microiontophoresis; N =6)are expressed as the percent of baseline diameter (100%). Data are means SE. a Significant difference (P < 0.05) when compared to the baseline diameter b Significantdifference when compared to the values of the juxtamedullary vessels

micropipette containing a 1 mM norepinephrine solution or aDimensions of juxtamedullary and cortical efferent arterioles pipette filled with norepinephrine-free saline. The drug-free The length and basal diameters of the renal arterioles from saline solution was used to test if the electrical pulses alonethe juxtamedullary and cortical glomeruli which met the func- could induce arteriolar constriction. The minimum diameters oftional criterion (at least 20% afferent arteriole constriction to 8 the arterioles were measured during each microiontophoresisHz) are presented in Table 1. The length of the juxtamedullary injection period. efferent arterioles, ranging from 1,250 to 3,100 m, were almost 10 times the length of the cortical efferent arterioles (ranging Dataanalysis from 170 to 365 gm). The average diameter of the juxtamedul- Controlvalues for arteriolar diameters and lengths werelary efferent arterioles (ranging from 12 to 29 jim) was almost calculated as mean SE. The minimum diameter during renal50% larger than cortical efferent vessels (ranging from 9 to 18 nerve stimulation or exposure to norepinephrine was thenjim). Juxtamedullary afferent arterioles (14 to 24 jim) were also calculated as a percent of the immediately preceding baselineconsiderably larger in diameter than cortical afferent vessels (9 diameter. to 17 jim) and often branched from the distal arcuate or The minimum arteriolar diameters during renal nerve stimu-proximal interlobular artery. The ratio of efferent:afferent di- lation (2, 4 and 8 Hz), or during exposure to norepinephrineameters was greater than 1.0 for juxtamedullary glomeruli but were compared against the baseline arteriolar diameter using awas equal to or less than 1.0 for cortical glomeruli. one-way analysis of variance with repeated measures followed by a Student-Newman-Keuls Test. Comparison of the frequen- Arteriolar responses to renal nerve stimulation cy-response curves obtained from outer cortical afferent and In every experiment, when the afferent arteriole of a jux- efferent arterioles, juxtamedullary afferent and efferent arteri-tamedullary glomerulus constricted to nerve stimulation, the oles (100, 500, and 1,000 m away from the glomeruli) wasefferent arteriole of the same glomerulus also constricted. The made by a two-way analysis of variance with repeated measuresarteriolar responses generally occurred within 5 seconds and followed by a Duncan's multiple-range test. In all statisticalreached a maximum constriction 20 seconds after stimulation. tests a probability less than 0.05 was considered significant. The recovery to baseline diameters generally was noted within 2 to 5 seconds and was complete within 20 to 30 seconds after Results terminating the stimulation. As illustrated in Figure 1, the juxtamedullary afferent and efferent arterioles (at sites within Glomerular selection 100 jim from glomerulus segment) constricted similarly to the In the initial experiments we first selected juxtamedullarythree stimulation frequencies. Furthermore, the more distal glomeruli with the largest and longest efferent arterioles, similarportions of the juxtamedullary efferent arterioles also con- to the average length of the efferent arterioles selected bystricted (Fig. 2); however, the degree of constriction decreased Steinhausen and colleagues [28, 291. But the afferent arteriole ofas the distance from the glomerulus increased. The segment only one of four of these glomeruli responded to nerve stimu-1,000 jim from the glomeruli constricted significantly less than lation. In contrast, other juxtamedullary glomeruli with efferentthe segment within 100 jim from the glomeruli. The afferent arterioles not as long and large were present in larger numbersarterioles of outer cortical glomeruli constricted by the same and approximately 80% of the afferent arterioles of theseextent as juxtamedullary afferent and efferent arterioles to glomeruli responded to nerve stimulation. Thus, after severalnerve stimulation (Fig. 1). However, the outer cortical efferent experiments these juxtamedullary glomeruli were selected first.arterioles of the same glomeruli did not respond. The percentage of outer cortical afferent arterioles (80%) which responded to nerve stimulation was similar to the percentage of Arteriolar responses to bath norepinephrine responding juxtamedullary afferent arterioles associated with The low bath concentration of norepinephrine (1 jiM) signif- glomeruli having shorter and smaller efferent arterioles. icantly constricted the outer cortical afferent arterioles but not Chen and Fleming: Neural control ofjuxiamedullary arterioles 687

100 100

90 80

a, a, a, a, aE 80 aE60 0 a, a, C C a, aa, a a 70 40 0 0

60 20

50 0 0 6 10 RNS NE(1 riM) NE(3 lIM) Stimulation frequency, Hz Fig. 3. Reduction in diameters (expressed as a percent of baseline JM-AFF)and Fig.1. Reduction in diameters (expressed as a percent of baselinediameter) of juxtamedullary afferent arterioles (, outer corti-juxtamedullary efferent arterioles (,JM-EFF)evoked by renal nerve diameter) of outer cortical afferent arterioles (A,C-AFF), stimulation (RNS) at 8 Hz, norepinephrine at bath concentration of I cal efferent arterioles (0,C-EFF),juxtamedullary afferent arterioles pM (NEJ M) and 3 p.M (NE3 p.M). Data are means SE from 5 rats. *J) (A, JM-AFF), and juxtamedullary efferent arterioles (I,JM-EFF) evoked by renal nerve stimulation (2,4,8 Hz). Data points are means <0.05, when compared against the baseline diameter (100%). SE from 6 rats.

100 norepinephrine bath concentration (3 PM). A comparison of the * diameter changes of outer cortical efferent versus juxtamedul- lary efferent arterioles to the high bath concentration revealed no significant difference. 90 Arteriolar responses to microiontophoretic injection of norepinephrine a, a, The local microinjection of 0.1 m norepinephrine con- aE 80 stricted the outer cortical afferent arteriole but not the efferent a, C vessels (Table 1, Fig. 4). The higher norepinephrine pipette aa, concentration (1 mM) caused the efferent arterioles to constrict a by 52% and the afferent arteriolar lumens to completely close. 0 70 Intense constriction of afferent arterioles usually occurred within 2 to 3 seconds after initiating the electrical pulses; in contrast the onset of efferent responses was generally slower, 60 requiring 5 to 10 seconds. No change in arteriolar diameters was observed when the pipette contained norepinephrine-free sa- line. Discussion 50 0 2 4 6 8 10 In this study we quantitated the responses ofjuxtamedullary (JM) afferent and efferent arterioles to brief renal nerve stimu- Stimulation frequency, Hz lation by directly visualizing the intact microcirculation of rat Fig. 2. Reduction in diameters (expressed as a percent of baseline JM- hydronephrotic kidneys. The JM glomeruli were distinguished diameter) of juxtamedullary efferent arterioles at 100 p.m (A, from outer cortical glomeruli by the long length of the efferent EFFJOO), 500 p.m (0,JM-EFF500),and 1,000 p.m (A,JM-EFFJ,000) away from the glomeruli evoked by renal nerve stimulation (2,4,8 Hz). arterioles [4, 11, 12, 19, 25]. After initially selecting a JM Data points are means SE from 8 rats. *Values of JM-EFFIOO were glomerulus based on the visual clarity of the vessel walls for significantly different from JM-EFFI ,000 values. precise diameter measurements, the renal nerve branch was stimulated. If the afferent arteriole of the selected glomerulus did not constrict by at least 20% to 8 Hz nerve stimulation, the juxtamedullary afferent arterioles or the efferent arteriolesanother JM glomerulus was selected. This criterion was applied of either glomerular type (Table 1, Fig. 3). At the higherto confirm that innervation to the selected glomerulus was norepinephrine concentration (3 MM), only the outer corticalintact. We reasoned that some glomeruli would not respond to efferent arterioles did not constrict significantly to the higher the electrical stimulation because they were not innervated by 688 Chen and Fleming:Neural control of juxta,nedullary arterioles

100 study. We cannot offer any definitive explanation for why the afferent arterioles did not respond to nerve stimulation among 75% of the JM glomeruli with large and long efferent arterioles, 80 or why 20% of the afferent arterioles of the other JM glomeruli and outer cortical glomeruli did not respond. Unresponsiveness ci) ci) of some outer and inner glomeruli to renal nerve stimulation E60 could relate to: (a) normal physiological variation among these

Cci> afferent vessels, (b) a selective disruption of the innervation to ci) some glcimeruli, (c) innervation by a different renal nerve 40 branch from the one being stimulated by our electrodes or, (d) 0 a physiological mechanism, such as greater endogenous vaso- dilator prostaglandin influence which opposed the constriction 20 of some afferent arterioles [24, 28]. It is important to emphasize that oUr data on afferent versus efferent arteriolar responses to nerve stimulation among outer and inner cortical glomeruli 0 were derived only from glomeruli with afferent arterioles re- sponding to nerve stimulation, and thus may not represent the ANS NE1 I.tM NE3tM NEO.1 msi NE1mi Bath responses of all glomeruli. Microlontophoresis The diameters of the JM afferent arterioles which met the FIg. 4.Reductionin diameters (expressed as a percent of baselineresponse criterion were significantly larger in diameter than the diameter) of outer cortical afferen: arterioles (,C-AFF)and corticalouter cortical afferent arterioles (Table 1), which is consistent efferent arterioles (,C-EFF,evoked by renal nerve stimulation (RNS) at 8 Hz, norepinephrine at bath concentration of I p (NE 1 pM) and with histological data from normal kidneys [4, 10, 34] and with 3 p" (NE 3 /sM,l, and microiontophoretic application of 0.1 mM (NE 0.1 two priOr studies of JM glomeruli in the rat hydronephrotic mM) and 1 mzci (NE 1 mM,) norepinephrine. Data are means sa from 6 kidney by Steinhausen and co-workers [28, 29]. The JM efferent aiS.*P<0.05, when compared against the baseline diameter. arterioles of our study were nearly 10 times longer than the cortical efferent arterioles (Table 1), yet they were considerably shorter than 4 of 11 JM efferent arterioles selected by the the renal nerve branch which was stimulated. Also, we consid-Steinhausen group [291. In all instances, when the afferent ered that the innervation of some glomeruli could have beenarteriole of a JM glomerulus constricted to nerve stimulation, damaged by the development of hydronephrosis or by thethe efferent arteriole also constricted (Fig. 1). Efferent arteriolar trauma of preparing the kidney for observation. When theconstriction occurred near the glomerulus (within 100 pm) and afferent arteriole of the selected glomerulus constricted to nerveas far as 1000 m downstream (Fig. 2). However, the degree of stimulation, the response of the éfferent arteriole of that sameconstriction diminished as the distance from the glomerulus glomerulus was always included in the data set based onincreased. This response pattern is consistent with the distri- evidence that the innervation of the efferent arteriole is anbution of vascular smooth muscle cells along the length of JM extention of nerves innervating the preglomerular vessels [12,efferent arterioles [10, 181. Outer cortical afferent arterioles 141. These same principles were applied to the selection of theconstricted to nerve stimulation in a fashion indistinguishable outer cortical glomerUli. from the JM vessels (Fig. 1). However, as we previously In the initial experiments, a small number of large JMobserved [13], the efferent arterioles associated with these same glomeruli (usually 2 to 3) with large and long efferent arteriolesouter cortical glomeruli failed to respond (Fig. 1). These data were apparent in the kidney tissue. These JM glômeruli werereveal a marked difference in the response of JM efferent versus selected because they were readily located and comparable toouter cortical efferent arterioles to renal nerve stimulation. the JM vessels studied previously by Steinhausen [28, 29].Despite a reduction in glomerular blood flow due to afferent However, the afferent arteriole of only on of four of thesearteriolar constriction, the equivalent constriction of the JM glomeruli constricted to renal nerve stimulation. Other moreefferent arterioles could maintain a higher glomerular filtration abundant JM glomeruli had shorter efferent arterioles (1,250 torate among JM nephrons than outer cortical glomeruli, where 3,000m)of slightly smaller diameter (Table 1) but were by allthe preglomerular vessels primarily constrict. The overall result anatomical criteria also JM glomeruli (the ratio of efferent:during episodes of increased efferent renal nerve activity would afferent arteriole diameters greater than 1, afferent arteriolesbe to sustain filtration among the JM glomeruli and to maintain often branched from proximal interlobular or distal arcuatethe medullary osmotic gradient necessary to preserve the artery [4]). The vast majority (approximately 80%) of theurinary concentrating mechanisms [36]. Inferences to normal afferent arterioles of these JM glomeruli responded to nervekidney function based on these microvascular observations stimulation, similar to the percentage of the afferent arteriolesfrom hydronephrotic kidneys must be made with caution. Yet, of outer cortical glomeruli which responded to renal nervethere are data from normal rat kidneys which are consistent stimulation. TherefoEe, because these JM glomeruli were con-with the notion that filtration among JM glomeruli is better siderably more prevalent in our kidneys and because themaintained than outer cortical glomerular filtration during renal percentage of responding afferent arterioles was comparable tonerve stimulation. In three studies, .Pelayo, Ziegler and Blantz the percentage of outer cortical affërent arterioles responding tomeasured single glomerular filtration rate (SNGFR) nerve stimulation, we preferentially selected the JM glomeruliamong superficial glomeruli and whole kidney GFR during renal with the shorter efferent arterioles for the remainder of thenerve stimulation. In the first study [22], the calculated vascular Chen and Fleming: Neuralcontrolof juxtamedullary arterioles 689 resistance changes for afferent and efferent vascular resistancesneurotransmitter of renal sympathetic nerves [2, 6]. In prepa- were comparable (t44%and 34%, respectively). The percentrations with intact renal microvascular networks [7, 17, 21, 27, change in SNGFR ( 24%) matched the decline in GFR (j28, 30, 32], outer cortical efferent arterioles are less reactive to 25%), indicating that if the inner cortical glomeruli respondednorepinephrine than outer cortical afferent arterioles. Whereas, differently to nerve stimulation, their impact on total kidneyisolated and individually perfused outer cortical efferent and GFR was insignificant. But in two other studies [23, 24]theafferent arterioles appear to be equally responsive to norepi- changes in SNGFR among the superficial glomeruli ( 26% andnephrine [11, 35]. Thus, we considered that the upstream 30%) were greater than the changes in GFR (118%and constriction of preglomerular vessels to norepinephrine, and 16%) during nerve stimulation. In these studies the increase inpossibly to nerve stimulation, could diminish the efferent arte- preglomerular resistance during nerve stimulation (187%andriolar response by an unknown mechanism. In our experiments, t107%)were at least twice the increase in postglomerularwhen norepinephrine was added to the bath (1 and 3 SM), the resistance ( '36%and 55%) among the superficial glomeruli.outer cortical efferent arterioles were less reactive than the One interpretation of these results which corroborates ourouter cortical afferent arterioles (Table 1), consistent with other direct microvascular observations is that when renal nervereports mentioned previously. But we considered that the stimulation evokes an greater constriction of afferent arteriolesconstriction of the interlobular and afferent arterioles evoked than efferent arterioles among the outer cortical nephrons, as iswith this route of administration could have diminished the usually reported [1, 13, 18, 23, 24, 31, 32], the decrease inresponse of the downstream efferent arterioles, not by a myo- SNGFR for superficial cortical glomeruli will be greater thangenie mechanism since efferent arteriolar contraction to norepi- the change in whole kidney GFR because the filtration ratenephrine seems to be independent of pressure within the vessel among the other glomeruli is not decreased to the same extent.[35], but by another mechanism, perhaps involving an endothe- But when the constriction of afferent and efferent arterioles ofhal cell product. By microinjecting norepinephrine around only outer cortical glomeruli is nearly equivalent, similar to thethe efferent arterioles, constriction of preglomerular vessels response pattern we are reporting among the inner corticalwas avoided. With this technique, the outer cortical efferent glomeruli, then the SNGFR will approximate the GFR. arterioles constricted (confirming their ability to respond to It is also important to recognize that the data of this studyintense adrenergic stimulation), yet they were still substantially address the acute microvascular alterations associated withless reactive than the afferent arterioles (Fig. 4). Thus, we brief nerve stimulation. With long-term increases in efferentspeculate that the outer cortical efferent arterioles do not renal nerve activity, local modulatory mechanisms are evokedconstrict to nerve stimulation because the amount of neurahly- [24] which may alter the preglomerular and/or postglomerularreleased norepinephrine is insufficient to activate the smooth vascular resistance changes of outer and inner glomeruli. muscle cells. There are no direct data to support this notion, In the second phase of this study, we addressed why theonly histological evidence suggesting that there are fewer outer cortical efferent arterioles in the hydronephrotic kidneynerves present around the outer cortical efferent vessels than preparations do not respond to nerve stimulation. Numerousafferent arterioles [3, 12]. investigations have reported greater elevation of preglomerular In assessing the reactivity of JM and outer cortical arterioles vascular resistance than postglomerular resistance [1, 18, 22—to norepinephrine bath (1 ELM), we noted that the constriction of 24, 31, 32] during renal nerve stimulation. In many, but not allouter cortical afferent arterioles (by 144%) was statistically [31] of these micropuncture studies, the postglomerular resis-more than inner cortical (JM) afferent constriction (Table 1). tance significantly increased. So why don't the outer corticalThis observation is consistent with a report by Steinhausen and efferent arterioles constrict in our experiments? Several impor-co-workers [28] that i.v. infusion of a low norepinephrine dose tant considerations have been proposed previously [13]. First,(sufficient to elevate blood pressure by only 14 mm Hg) con- we consider the duration of the nerve stimulation period to bestricted the outer cortical afferent arterioles (by 16 2%) but an important factor (30 seconds in our experiments compared tonot the JM afferent arterioles (by 4 3%). Thus, both reports as long as 45 minutes in the micropuncture experiments [22—indicate that the afferent arterioles of outer cortical glomeruhi 24]). With the long stimulation periods, there is sufficient timeare more reactive to a low level of norepinephrine than afferent for angiotensin II levels to increase in the kidney as a result ofvessels of the inner cortex. In our experiments, the disparity neurally-mediated release. Indeed, Pelayo and colleaguesbetween outer and inner cortical afferent arteriole reactivity [22] have demonstrated that angiotensin II is a crucial compo-was not observed at a higher norepinephrine bath concentration nent of the postglomerular vascular resistance change calcu-(3 LM, Table 1). The Steinhausen group did not assess arteriolar lated from micropuncture data. In our experiments, the dura-reactivity to a higher norepinephrine dose but Wilson [34] tion of nerve stimulation was limited to 30 seconds to minimizeutilized perfusion-fixation techniques to quantitate the diameter the role of these non-neural factors in the arteriolar responses.of afferent arterioles in normal kidneys following norepineph- Since roughly 75% of the maximum arteriolar response ofrine admininstration. Similar to our direct observations, he preglomerular arterioles was achieved within 5 seconds afterreported that outer and mid cortical afferent arterioles con- stimulating the nerve and since the arteriolar diameters recov-stricted to the same extent as juxtamedullary afferent vessels to ered to 80% within 10 seconds after terminating the stimulationa norepinephrine dose sufficient to elevate blood pressure by 50 [131, the responses appear to be neurogenic. mm Hg. Thus, with a high level of adrenergic receptor stimu- In this study we focused on one possible explanation for thelation afferent arterioles at all cortical levels appear to respond unresponsiveness of the outer cortical efferent arterioles toequally. renal nerve stimulation; that is, the outer cortical efferent The constriction of efferent arterioles to a low dose of i.v. vessels are relatively insensitive to norepinephrine, the primarynorepinephrine [28] or a low bath norepinephrine concentration 690 Chen and Fleming:Neural control offuxtamedullary arterioles

(1 m, Table 1) was modest and comparable between outer and 11. EDWARDS RM: Segmental effects of norepinephrine and angioten- inner cortical efferent arterioles. With high norepinephrine sin II on isolated renal microvessels. Am J Physiol 244 (Renal Fluid levels, a greater constriction was elicited but still no distinction Electrol Physiol 13):F526—F534, 1983 12. FOURMAN J: The adrenergic innervation of the efferent arterioles between outer versus inner cortical efferent responses was and the in the mammalian kidney. Experientia 26:293— found (Table 1). Hence, there is no evidence that efferent 294, 1969 arterioles of outer cortical glomeruli respond differently from 13. FLEMINGJT,Z-IANG C, CHEN J, PORTER JP: Selective preglomer- inner cortical arterioles at low or high levels of adrenergic ular constriction to nerve stimulation in rat hydronephrotic kid- receptor stimulation with norepinephrine. Yet, to renal nerve neys. Am J Physiol 262 (Renal Fluid Electrol Physiol 31): F348— F353, 1992 stimulation the inner cortical efferent arterioles are responsive 14. GORGAS K: Innervation of the juxtaglomertilar apparatus, in Pe- while the outer cortical efferent arterioles are unresponsive. ripheral Neuroendocrine Interaction, edited by COUPLAND RE, This disparity highlights the potential difference in alpha adren- FORSSMANN, WF. New York, Springer-Verlag, 1978, pp. 144—152 ergic receptor activation by norepinephrine versus activation 15. GOTSHALL RW, ITsKovrrs HD: Redistribution of renal cortical by neural stimulation. blood flow by renal nerve stimulation and norepinephrine infusion. In summary, in rat hydronephrotic kidneys, when the afferent Proc Soc Exp Biol Med 154:60—64, 1977 16. HERMANSSONK,LARSON M, KALLSKOG 0, WOLGAST M: Influ- arterioles of juxtamedullary glomeruli constrict to renal nerve ence of renal nerve activity on arteriolar resistance, stimulation the efferent arterioles also constrict by the same and fluid reabsorption. Pfluigers Arch 389:85—90, 1981 extent. This differs from the preferential constriction of afferent 17. KIMURA K, HIRATA Y, NANBA S, Toso A, MATSUOKA H, SUGIM- arterioles among outer cortical glomeruli. The lack of constric- OTO T: Effects of atrial natriuretic peptide on renal arterioles: Morphometric analysis using microvascular casts. Am J Physiol tion by cortical efferent arterioles to sympathetic nerve stimu- 259 (Renal Fluid Electrol Physiol 28):F936—F944, 1990 lation does not appear to be associated with the constriction of18. KON V, IcHIKAwA I: Effector loci for renal nerve control of cortical upstream preglomerular vessels but to their inherent insensitiv- microcirculation. Am J Physiol 245 (Renal Fluid Electrol Physiol ity to norepinephrine. These observations, which should be 14):F545—F553, 1983 cautiously extrapolated to normal kidneys, reveal that sympa-19. KRIZ W: Structural organization of the : Comparative thetic neural control of glomerular hemodynamics may be and functional aspects. Am J Physiol 241:R3—R16, 1981 20. LJUNGQVIST A, WAGERMARK I: The adrenergic innervation of different between outer cortical and juxtamedullary glomeruli. intrarenal glomerular and extra-glomerular circulatory routes. Nephron 7:218—229, 1970 Acknowledgments 21. LOUTZENHISER R, HAYASIH K, EPSTEIN M: Atrial natriuretic peptide reverses afferent arteriolar vasoconstriction and potentiates This research was supported by a grant from the National Institutes efferent arteriolar vasoconstriction in the isolated perfused rat of Health, Heart, Lung and Blood Institute (HL42447). kidney. J Pharmacol Exp Ther 246:522—528, 1988 22. 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