We investigated the role of the inward rectifier K+ channel (KIR) in the renal interlobular artery (ILA). The ILA supplies the afferent arteriole and ranges in diameter from >100 µm near its origin at the arcuate artery to <30 µm at its most distal segment.
Vasodilatory responses to elevated extracellular K+ (15 mmol/L) and vasoconstrictor responses due to KIR blockade by Ba2+ (10–100 µmol/L) were assessed in in vitro perfused hydronephrotic rat kidneys. The distal ILA (26 ± 1 µm) exhibited K+-induced dilation and Ba2+-induced vasoconstriction, whereas neither response was observed in the proximal ILA (108 ± 3 µm). The intermediate ILA (55 ± 1 µm) exhibited a modest K+-induced vasodilatation, but no Ba2+-induced vasoconstriction. The K+-induced dilations were blocked by Ba2+, but not by ouabain. Ba2+-induced depolarization, measured in ILA segments from normal kidneys, decreased with the increasing diameter. Patch-clamp studies demonstrated that the KIR current (IKIR) density also was inversely correlated with ILA segment diameter. Myocytes from afferent arterioles and distal ILAs exhibited similarly large IKIR, whereas this current was absent in proximal ILA myocytes. Finally, we found that Ba2+ attenuated myogenic vasoconstriction, suggesting an involvement of IKIR. The previously shown pattern of myogenic reactivity of the ILA (distal > intermediate > proximal) mirrors the distribution of IKIR reported in the present study, further supporting a role for IKIR.
Our findings indicate differences in the magnitude of IKIR along the ILA and suggest that the influence of KIR on reactivity increases as vessel diameter decreases from proximal to distal regions.
The inward rectifier (KIR) occupies a unique niche among vascular K+ channels. These channels typically do not contribute to the membrane potential of larger conduit arteries, but are commonly expressed in resistance vessels, suggesting an important role in the regulation of blood flow and microvascular pressures. Moreover, KIR is implicated in responses unique to resistance vessels. For example, elevations in interstitial K+ elicit vasodilatation, in part, by modulating KIR1–3 and this mechanism has been suggested to contribute to responses attributed to an endothelium-derived hyperpolarizing factor (EDHF).4 KIR has been implicated in myogenic vasoconstriction and in the conduction of electrical signals along small arterioles, as membrane stress inhibits KIR in cerebral vessels5 and blockade of KIR blocks conducted vasodilation.6–8 Accordingly, information on the distribution of this channel is important in regard to our understanding of the integration of signals within the vasculature and the functional properties of the various segments of the vasculature.
Regional heterogeneity in the distribution of KIR is seen in a number of vascular beds9,10 and may also exist within the kidney. Prior et al.11 demonstrated that the arcuate artery, a renal conduit vessel, does not exhibit Ba2+-sensitive K+-induced vasodilatation and, while KIR could be detected in a fraction of arcuate myocytes (∼5%), the current density was too low to play a significant role in regulating membrane potential. In contrast, our laboratory found KIR to be a major determinant of membrane potential in the renal afferent arteriole12 and that myocytes isolated from pre-glomerular afferent and post-glomerular efferent arterioles express KIR2.1 and exhibit Ba2+-sensitive KIR currents.13 Similarly, Cao et al.14 found KIR to be present in the smooth muscle-like contractile pericytes that regulate the diameter of the descending vasa recta. The interlobular artery (ILA, also known as the cortical radial artery) originates at the arcuate artery and radiates towards the surface of the kidney, terminating with the formation of the afferent arterioles. The ILA exhibits differing properties in the larger proximal vs. smaller distal segments. For example, while the distal ILA exhibits myogenic vasoconstriction, this property is not seen in the larger proximal segments.15,16 The function of KIR has not previously been examined in this unique vessel, which exhibits properties of both conduit and resistance vessels along its length.
To address this issue, we studied the functional role and the distribution of KIR along the ILA, using the in vitro perfused hydronephrotic rat kidney model and patch-clamp techniques. Vasoconstrictor responses to Ba2+ and vasodilatory responses to increases in external K+ ([K+]o) were evaluated in proximal, intermediate, and distal segments of the ILA using the hydronephrotic kidney. Whole-cell voltage-clamp studies were used to evaluate KIR currents in native myocytes obtained from afferent arterioles and proximal, intermediate, and distal segments of the ILA individually isolated from the normal rat kidneys. The perforated-patch technique was used to measure changes in membrane potential in individual ILA segments. Our findings reveal segment-specific variation in the role of KIR along the ILA and have important implications regarding the determinants of vasomotor regulation in the renal vasculature.
All procedures complied with University of Calgary Animal Ethics and Canadian Council on Animal Care regulations and conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The in vitro perfused hydronephrotic rat kidney was used to evaluate the effects of Ba2+ and elevated [K+]o on the ILA. Past studies demonstrate that responses in this model correspond to not only those of vessels isolated from normal kidneys, but also to the responses of normal kidneys in vitro and in vivo.13,17–20 Unilateral hydronephrosis was induced in male Sprague–Dawley rats by ligating the left ureter under halothane anaesthesia. Hydronephrotic kidneys were harvested, using halothane anaesthesia, after 6–8 weeks when advanced tubular atrophy allows visualization of the microvasculature. In each case, the level of anaesthesia was monitored by assessing digital reflex responses. The renal artery was cannulated and the kidney excised with continuous perfusion. Perfusion pressure was monitored at the renal artery. Kidneys were allowed 1 h to recover prior to initiation of experimental protocols. Diameters were measured by online image processing.12,13,17,20 Hydronephrotic kidneys were perfused with modified Dulbecco's minimum Eagle's medium (GIBCO) containing (in mmol/L) 1.6 Ca2+, 30 bicarbonate, 5 glucose, 1 pyruvate and 5 HEPES. Ibuprofen (10 µmol/L) was added to eliminate the effects of renal prostanoids.20 Elevated KCl solutions were prepared by isotonic substitution for NaCl. Ouabain was prepared fresh for each experiment (3 mmol/L). Phentolamine and propranolol (10 µmol/L) were added to the ouabain solution to avoid effects mediated by transmitter release.
KIR currents (IKIR) were evaluated using patch clamp (23°C). Renal microvessels were isolated from the renal cortex (excluding the juxtamedullary area) of normal rat kidneys using the gel-perfusion technique.19 Diameters were estimated by a calibrated ocular reticle and ILA segments ranging from 30 to 70 μm were selected for the study. Voltage-clamp studies were performed using the whole-cell configuration on native myocytes obtained from individual microvessels.13 Borosilicate glass pipettes were pulled to a 2–3 µm tip diameter and fire polished. Pipettes were filled with a solution containing (mmol/L): K+-gluconate (120), KCl (20), HEPES (10), Na2ATP (2), Na2GTP (0.1), EGTA (5), MgCl2 (2), CaCl2 (0.1), pH 7.2, and pipette resistance 6–12 MΩ. The free Ca2+ and Mg2+ were calculated to be 5.1 nmol/L and 0.38 mmol/L (Maxchelator, http://www.stanford.edu/~cpatton/downloads.htm). The 30 mM K+ external bath solution contained (mmol/L): KCl (30), NaCl (110), MgSO4 (1), HEPES (5), CaCl2, (1.5), and glucose (5.6), pH 7.35. To minimize contributions of other K+ channels, 5 mmol/L 4-aminopyridine and 10 µmol/L glibenclamide were included in the bath solution. In whole-cell configuration, a voltage-ramp protocol (−120 to +60 mV, 1 s) was applied every 5 s from a holding potential of −40 mV (pClamp, v.8). Currents were digitized at 500 µs intervals and filtered with low-pass filter at 2 KHz online (Axoclamp 200B amplifier; Axon Instruments, Union City, CA, USA). Traces were outputted at 1 KHz off-line for analysis. An agar bridge (3 mol/L KCl) was used with the reference electrode. Data were not corrected for the liquid junction potential (∼4 mV).21 Membrane potentials were recorded in arterioles (32°C), using the perforated-patch method with 400 µg/ml nystatin in a pipette solution containing (mmol/L): K+-gluconate (120), KCl (20), HEPES (10), EGTA (5), MgCl2 (0.5), and CaCl2 (0.1), pH 7.2.
Data are expressed as the means using the standard error of the mean as an index of dispersion. Differences between means were evaluated by paired or unpaired Student's t-test. Probabilities (P) of <0.05 were considered significant. For multiple measurements, analysis of variance (ANOVA) followed by the Bonferroni t-test was applied to assess significance.
To evaluate the functional role of KIR in intact vessels, we determined whether elevated extracellular K+ evoked a Ba2+-sensitive and ouabain-insensitive vasodilatation and whether blockade of KIR by Ba2+ induced a vasoconstriction. Since the ILA is tapered along its length, segments were identified based on the basal diameter.15 ILAs were selected near their origin (proximal ILA diameter 107.5 ± 2.9 µm, n= 6), at an intermediate point (intermediate ILA diameter 52.4 ± 1.1 µm, n= 5), or at their termini (distal ILA diameter 26.2 ± 0.9 µm, n= 26).
K+-induced vasodilatation in the distal ILA
Figure 1A illustrates the effects of elevating [K+]o on the myogenic vasoconstriction in a 21 µm (distal) segment of an ILA. Note that the vasoconstriction induced by increasing renal arterial pressure (RAP) from 80 to 160 mmHg and the vasodilation evoked by increasing [K+]o (15 mmol/L). Returning [K+]o to 5 mmol/L restored the vasoconstriction. Mean data are presented in Figure 1B. Increasing RAP reduced diameters from 26.2 ± 0.9 to 17.5 ± 1.0 µm (n= 26) and 15 mmol/L KCl increased diameters to 26.3 ± 1.2 µm (P< 0.0001). In a separate series, pressure was held at 80 mmHg and tone was elevated with angiotensin II (Ang II, 0.1 nmol/L). Diameters were reduced from 21.9 ± 0.4 to 8.2 ± 0.8 µm (P= 0.001, n= 4; Figure 1B) and 15 mmol/L [K+]o increased diameters to 21.2 ± 0.8 µm (P= 0.001 vs. Ang II alone). Returning [K+]o to 5 mmol/L restored the Ang II-induced vasoconstriction (7.9 ± 0.9 µm, data not shown).
Elevations in [K+]o may evoke hyperpolarization and vasodilatation by enhancing the outward component of IKIR or by stimulating the electrogenic Na+/K+ ATPase pump.1,3,11,22 To evaluate the contributions of these two mechanisms, we determined the effects of Ba2+ (100 µM) and ouabain (3 mmol/L) on the vasodilatation evoked by elevated [K+]o. In these studies, the administration of Ba2+ or ouabain was sufficient to establish a vasoconstriction. As shown in Figure 1B, Ba2+ decreased distal ILA diameters from 21.5 ± 1.0 to 8.9 ± 0.7 µm (n= 6) and the subsequent administration of 15 mmol/L KCl failed to evoke vasodilatation (8.6 ± 0.6 µm, P= 0.38). Ouabain reduced distal ILA diameters from 21.6 ± 0.9 to 7.9 ± 0.7 µm (n= 11; Figure 1B). As described previously,12 to compensate for the effects of ouabain on intracellular [K+], extracellular [K+] was increased from 5 to10 mmol/L in these experiments. This manipulation increased diameters to 15.5 ± 1.2 µm (P< 0.0001). In a separate series, distal segments were treated with a combination of ouabain plus 100 µmol/L Ba2+, which reduced diameters to 8.5 ± 0.7 µm. The subsequent addition of 10 mmol/L KCl failed to significantly increase diameter (7.6 ± 0.6 µm, P= 0.06, n= 8). Thus K+-induced vasodilatation of the distal ILA was inhibited by Ba2+, but not by ouabain, implicating the involvement of KIR.
K+-induced responses of the intermediate and proximal ILA
The intermediate ILA has less myogenic reactivity and the proximal ILA does not exhibit this response.15,16 For this reason, Ang II (1 nmol/L) was used to establish basal tone for these studies. Ang II reduced diameters of the intermediate ILA from 55.4 ± 1.4 to 43.2 ± 1.2 µm (Figure 2A, n= 5). Increasing [K+]o to 15 mmol/L increased diameters to 47.0 ± 1.5 µm (P= 0.0034). Diameters recovered to 42.5 ± 0.8 µm when [K+]o was returned to 5 mmol/L (data not shown). Ouabain, administered in the presence of Ang II, had no additional effect on diameter (42.7 ± 0.9 µm), and elevating [K+]o (10 mmol/L) increased diameters to 47.8 ± 1.7 µm (P= 0.006, Figure 2A, P= 0.18 vs. control). In contrast, Ba2+ inhibited K+-induced vasodilation. ILA diameters were 46.7 ± 1.6 µm in Ba2+ plus Ang II and 40.3 ± 2.8 upon addition of 15 mmol/L [K+]o (P= 0.14; Figure 2A). In a separate group treated with Ba2+, ouabain, and Ang II, diameters were 44.4 ± 1.6 µm in 5 mmol/L [K+]o and 38.7 ± 2.9 µm (P= 0.07) in 15 mmol/L [K+]o.
In the proximal ILA, 1 nmol/L Ang II reduced diameters from 107.5 ± 2.9 to 74.8 ± 6.4 µm (n= 6; Figure 2B). In contrast to the smaller ILA segments, rather than inducing vasodilatation, 15 mmol/L KCl caused a further constriction to 55.6 ± 4.6 µm (n= 6, P= 0.007). Figure 2C compares the responses of the distal, intermediate, and proximal ILA to elevated [K+]o. Vessel diameters are normalized to the basal diameter. Note that while a relatively modest K+-induced dilation was observed in the intermediate ILA, this response was absent in the proximal ILA. For comparative purposes, previously published data13 on afferent arteriolar response to elevated [K+]o in the presence of Ang II-induced vasoconstriction are also presented.
Segmental variations in the vasoconstrictor effects of Ba2+ on the ILA
We next determined whether blockade of KIR evokes vasoconstriction in the ILA. These studies were conducted at an RAP of 40 mmHg. Under these conditions, proximal ILA diameters were 100.8 ± 2.1 µm (n= 9; Figure 3A). Following the application of 10, 30, and 100 µmol/L Ba2+, diameters were 94.5 ± 5.2 µm (P= 0.26), 91.7 ± 7.6 µm (P= 0.27) and 89.4 ± 7.7 µm (P= 0.17), respectively. Similarly, Ba2+ did not constrict the intermediate ILA. Basal diameters were 51.1 ± 1.1 µm (n= 5) and diameters were 51.1 ± 1.5, 50.3 ± 1.2 µm (P= 0.2739), and 49.2 ± 2.1 µm (P= 0.2656) in 10, 30, and 100 µmol/L Ba2+, respectively (Figure 3A). In contrast, Ba2+ elicited a concentration-dependent constriction of the distal ILA. Diameters were 20.1 ± 0.9 µm in controls and 18.8 ± 1.2 µm in the presence of 10 µmol/L Ba2+ (n= 6, P= 0.26; Figure 3C). At 30 and 100 µmol/L Ba2+, diameters were reduced to 15.3 ± 1.4 µm (P= 0.0286) and 12.9 ± 1.3 µm (P= 0.0154), respectively (Figure 3A). Data summarizing the effects of Ba2+ on each ILA segment and, for comparative purposes, on the afferent arteriole under similar conditions (previously published12), are shown in Figure 3B. Diameters are normalized to the basal values for each vessel.
Figure 3C and D illustrates the differing effects of Ba2+ on membrane potentials of isolated vessels. As shown in the tracings (Figure 3C), 100 µmol/L Ba2+ evoked depolarization in the afferent arteriole, but not in a 60 µm ILA segment. Figure 3D illustrates the significant inverse relationship between the magnitude of the Ba2+-induced depolarization and diameter in nine vessels studied using this approach.
Patch-clamp studies of myocytes from afferent arterioles and segments of the ILA
The above observations suggest an important contribution of the outward component of IKIR in setting membrane potential of the afferent arteriole and distal ILA. Supplementary material online, Figure S1, illustrates the presence of the small outward IKIR seen in afferent arteriolar myocytes at −40 to −60 mV and the larger inward current seen at voltages negative to the K+ equilibrium potential (EK). This figure and Figures 4 and 5 also illustrate the effects of changing [K+]o from 5 to 30 mmol/L on the reversal potential of this current. Note the positive shift that is evoked by 30 mmol/L [K+]o , as is predicted by the effects on EK. To facilitate a quantification of IKIR in the various vascular segments, the magnitude of the inward component of this current was assessed in both physiological [K+]o (5 mmol/L) and in 30 mmol/L [K+]o.
Figure 4 (top) depicts images of ILA segments isolated using our approach and images of ILA myocytes obtained from each segment. The lower panel of Figure 4A–C depicts the current tracings from myocytes isolated from ILA segments of 30, 50, and 70 µm diameter, respectively. Currents were recorded in 5 and 30 mmol/L [K+]o and in the presence and absence of 100 µmol/L Ba2+. Note that myocytes from the most distal ILA segment (30 µm, lower left) exhibited discernable Ba2+-sensitive IKIR even in 5 mmol/L [K+]o, whereas this current could not be detected in myocytes from the larger ILA segments under these conditions. In 30 mmol/L [K+]o , IKIR was detected in both distal and intermediate (50 µm, lower centre) ILA myocytes, but not in myocytes from the proximal (70 µm) ILA.
Mean data illustrating the current–voltage relationship of the Ba2+-sensitive IKIR are depicted in Figure 5A and B. Myocytes isolated from afferent arterioles (20 µm) and from 30 µm distal ILAs exhibited clearly discernible IKIR in 5 mmol/L [K+]o [−8 ± 2 pA/pF, n= 10, and −11 ± 3 pA/pF, n= 4, respectively, measured at −120 mV(Figure 5A)]. A smaller current was detected in myocytes from 40 µm ILA in 5 mmol/L [K+]o (−4 ± 2 pA/pF, n= 6). In each case, IKIR was enhanced approximately five-fold in 30 mmol/L [K+]o. In 30 mmol/L [K+]o (Figure 5B), myocytes from afferent arterioles, 30, and 40 µm ILAs exhibited IKIR of −49 ± 11, −46 ± 17, and −20 ± 6 pA/pF (at −120 mV), respectively. In 30 mmol/L [K+]o, IKIR was also detected in myocytes from the 50 µm ILA segment (−8 ± 4 pA/pF, at −120 mV, n= 7). In this vessel, however, IKIR was not discernable in 5 mmol/L [K+]o (−0.7 ± 0.6 pA/pF, n= 7). In contrast, IKIR was not detectable in the myocytes from the 70 µm ILA segment in either 5 or 30 mmol/L [K+]o (4 ± 3 pA/pF, n= 5, and −2 ± 2 pA/pF, n= 6, respectively). Thus while it is not possible to rule out a contribution of endothelial IKIR in regard to the functional responses, the distribution of IKIR in the myocytes from the vascular segments corresponds closely with functional responses to Ba2+ and elevated [K+]o.
The relationship between the density of IKIR and vessel diameter is illustrated in Figure 5C. Note the inverse relationship between IKIR density (measured at −120 mV in 30 mmol/L [K+]o) and vessel diameter (P< 0.0001, correlation coefficient of −0.63). As shown in Figure 5D, while vessel diameter was related to current density, myocyte capacitance was not. As noted previously,13,19 the myocytes of the afferent arteriole are quite small. Myocytes isolated from the ILA were of similarly size, ranging in capacitance from 6 to 8 pF.
Effects of Ba2+ on myogenic response
As discussed below, the distribution of IKIR along the ILA corresponds to regional differences in the functional properties of this vessel, including myogenic reactivity.15 We therefore were interested in determining whether blockade of IKIR, by Ba2+, might alter myogenic responses. Our previous attempt to address this issue was thwarted by the Ba2+-induced vasoconstriction which prevented evaluations of myogenic reactivity in the presence of IKIR blockade.12 The protocol depicted in Figure 6A illustrates how we approached this issue in the present study. Myogenic reactivity was initially assessed (control), by recording steady-state diameters as RAP was increased from 80 to 180 mmHg, in 20 mmHg steps (1 min duration). As shown, this elicited a graded, pressure-dependent vasoconstriction. The kidneys were then treated with either 100 µmol/L BaCl2 or 0.1 nmol/L Ang II, reducing diameters from 16.9 ± 0.4 to 6.0 ± 0.4 µm (n= 5) and from 17.9 ± 0.8 to 6.2 ± 0.7 µm (n= 5), respectively. Pinacidil (1 µmol/L) was then administered and increased diameters to 14.7 ± 0.3 µm and 13.8 ± 1.4 µm, respectively. Myogenic reactivity was then reassessed under these conditions. The results are summarized in Figure 6B. Diameters were normalized to the per cent of the (basal) diameters at 80 mmHg to facilitate comparisons. In the Ang II group, basal diameters were 18.3 ± 0.7 µm in controls and 13.4 ± 1.4 µm following treatment with Ang II and pinacidil. Increasing perfusion pressures elicited similar decreases in diameters. At 180 mmHg, diameters were 8.0 ± 0.6 µm (44 ± 4 % of basal) in controls and 5.1 ± 0.6 µm (38 ± 5 % of basal) following treatment with Ang II and pinacidil (P= 0.09). In the group treated with Ba2+, basal diameters were 17.6 ± 0.5 and 14.7 ± 0.3 µm for controls and Ba2+ plus pinacidil. Myogenic responses were significantly reduced at all pressures. At 180 mmHg, diameters were 8.2 ± 0.9 µm (46 ± 4 % of basal) in controls and 12.2 ± 0.9 µm (82 ± 5 % of basal) following treatment with Ba2+ and pinacidil (P= 0.002).
A characteristic of KIR is that this channel is commonly seen in resistance vessels, but rarely observed in larger conduit arteries.9,10 The diameter of the ILA decreases as the vessel courses from its origin at the arcuate artery to the superficial regions of the cortex. The properties of the ILA change from that of a conduit vessel near its origin to that of a resistance vessel at its terminal segments. For example, the distal, but not proximal, segments exhibit myogenic vasoconstriction15,16 and intraluminal pressures are reduced only at the terminal portions of the ILA.23,24 In the present study, myocytes from the distal ILA exhibited large IKIR and this ILA segment responded to manipulations known to alter IKIR. This current was lacking in the proximal ILA, whereas the intermediate ILA exhibited mixed characteristics in regard to the function of KIR, consistent with a transition between a conduit and resistance vessel at this segment.
Ba2+ blocks IKIR in a relatively selective manner at concentrations below 100 µmol/L.9 We previously reported that 30 µM Ba2+ elicited depolarization and vasoconstriction in the afferent arteriole.12 Elevating [K+]o (from 5 to 15 mmol/L) induced a hyperpolarization in afferent arterioles preconstricted with Ang II and elicited a vasodilation that is blocked by Ba2+, but not by ouabain.12,13 We also reported IKIR in myocytes from afferent and efferent arterioles and both vessels expressed KIR2.1,13 the isoform implicated in K+-induced hyperpolarization in cerebral arteries.25 Increases in [K+]o shift the K+ Nernst potential to more positive values, accounting for the observed depolarization and vasoconstriction. Modest increases in [K+]o also increase the macroscopic current carried by KIR26 (Supplementary material online, Figure S1). The underlying mechanism involves a modification of the channel block by intracellular polyamine and/or Mg2+, which also accounts for the inward rectification.27,28 Accordingly, an elevation in [K+]o at the outer vestibule of the channel enhances the outward current. If IKIR contributes to membrane potential, modest increases in [K+]o evoke hyperpolarization and vasodilation in depolarized or pre-constricted vessels.13,22,29–31 Otherwise this manoeuvre enhances depolarization, facilitating vasoconstriction. Elevated [K+]o may also cause hyperpolarization by stimulating the electrogenic Na+/K+ ATPase.1,3,11 These two mechanisms are distinguished by their sensitivities to Ba2+ and ouabain. We did not determine whether the contribution of this latter mechanism varies along the ILA, but rather employed ouabain to rule out an involvement of Na+/K+ ATPase in the Ba2+-sensitive K+-induced vasodilation.
Ba2+ elicited a vasoconstriction in the distal ILA that was similar to that previously reported for the afferent arteriole (Figure 3). The distal ILA also dilated in response to elevated [K+]o in a manner comparable to that of the afferent arteriole12,13 (Figure 2C), although membrane potential responses were not obtained in the present study. The density of IKIR , however, was similar in myocytes from afferent arterioles and 30 µm ILA in both 5 and 30 mmol/L [K+]o (Figure 5). In contrast, the proximal ILA did not exhibit a detectable IKIR and constricted, rather than dilated, in response to elevated [K+]o (Figure 2B). Although Ba2+-induced vasoconstriction was not observed in either the intermediate or proximal ILA, the intermediate ILA exhibited a Ba2+-sensitive K+-induced vasodilatation. This vessel also exhibited a modest, but clearly detectable, IKIR in 30 mmol/L [K+]o (Figures 4 and 5). Consistent with the contractile responses, we observed that the magnitude of the Ba2+-induced membrane depolarization correlated inversely with vessel diameters, providing the direct evidence for the important role of IKIR in the control of membrane potential in the intact distal ILA.
Our analysis also demonstrated a significant inverse relationship between IKIR density and vessel diameter (Figure 5). A similar relationship has been reported in the coronary and cerebral vasculatures.26,31,32 Likewise, in the cremaster microcirculation, Ba2+-induced vasoconstriction is limited to forth-order (∼20 µm) arterioles.30 Prior et al.11 had shown that IKIR plays little role in the arcuate artery, from which the proximal ILA originates. The present study demonstrates that KIR current density increases with decreasing diameter along the ILA. In the most distal ILA segment, IKIR is similar in magnitude to that of the afferent arteriole. KIR is also expressed in the post-glomerular circulation and contributes to the regulation of membrane potential in the efferent arterioles13 and in the vascular pericytes of the descending vasa recta.14
The magnitude of IKIR observed in renal microvascular myocytes merits comment. Myocytes from afferent arterioles and distal ILA exhibited IKIR of −8 to −11 pA/pF in 5 mmol/L K+ and −46 ± 17 to −49 ± 11 pA/pF in 30 mmol/L K+ (measured at −120 mV), values relatively larger than those reported for other vascular myocytes.1,5,26,33 Although the magnitude of IKIR suggests a functional importance, the precise role of this current in the renal vasculature is not known. Alterations in IKIR, mediated by protein kinase C, are implicated in vasoconstrictor responses5,33,34 and this possibility requires additional study in the kidney. The significance of K+-induced vasodilation, while appreciated in the cerebral and muscle circulations,2 is not clear in renal circulation. It has been suggested that an efflux of K+ ions via endothelial K+ channels might constitute the long sought EDHF, via effects on KIR.4 This does not appear to be the case, however, for the afferent arteriole.35 The findings presented in Figure 6 suggest an involvement of KIR in the renal myogenic response, but the nature of this involvement is not apparent. For example, evidence suggests that IKIR acts to amplify vascular electrical signals8 and IKIR could play a permissive role in pressure-induced depolarization. KIR is also required for the transmission of signals along the vasculature6–8 and could be involved in distributing pressure signals within the renal circulation. The potential involvement of KIR in the renal myogenic response is of considerable interest. In this regard, it is noteworthy that the distribution of IKIR along the ILA corresponds closely to differences in myogenic reactivity along this vessel.15 Clearly, further investigations are required to elucidate the precise physiological roles of KIR in the renal microcirculation.
Conflict of interest: none declared.
This work was supported by the Canadian Institutes for Health Research (MOP-14075); the Heart and Stroke Foundation of Alberta, Intuit and N.W.T. and the Alberta Heritage Foundation for Medical Research (AHFMR; 20090234 and 20091182). S.V.S. was supported by a visiting scientist grant from AHFMR and an international travel grant from the Royal Society. R.L. is an AHFMR Senior Investigator. L.C. was an AHFMR doctoral student and an Honorary Killam Memorial Scholar.