INTRODUCTION

Top Abstract Introduction Methods Results Discussion References

RENAL BLOOD FLOW, glomerular filtration rate, and glomerular capillary pressure (P_GC) are precisely regulated over a broad range of renal perfusion pressures. This renal autoregulatory response is mediated by a vascular reflex coupled to distal tubular flow that is unique to the kidney (tubuloglomerular feedback, TGF) and by a direct, pressure-induced vasoconstriction of the afferent arteriole. The ability of the kidney to maintain a constant P_GC when perfusion pressure is increased is important not only for maintenance of normal renal function but also by serving to prevent glomerular hypertension and the associated glomerular injury. A disruption in P_GC regulation and the resultant glomerular hyperfiltration are considered to be primary factors in the pathogenesis of glomerular nephropathy and progressive renal disease (28). The reactivity of the afferent arteriole to pressure is altered by pathophysiological (6, 8) and physiological (24) factors. Unfortunately, we know little of the mechanisms regulating myogenic vasoconstriction in this vessel.

In the present study, we examined the effects of altering PKC activity on the afferent arteriolar response to pressure. PKC has been implicated in myogenic vasoconstriction in other vascular beds (25). However, the role of PKC in the regulation of renal afferent arteriolar myogenic reactivity has not been examined. The myogenic response of this vessel to elevated pressure differs in some regards from stretch-induced responses observed in larger resistance arteries. The myogenic component of renal autoregulation operates at 0.1-0.3 Hz both in the intact kidney in vivo (11) and in our in vitro model (4). The in vitro constrictor responses of the afferent arteriole to stepped increases in pressure reach steady state within 5-10 s and are initiated in the absence of a preceding increase in diameter (Fig. 1), differing in this regard from myogenic responses that are initiated by stretch. Because myogenic activation of the afferent arteriole is uniquely coupled to control of downstream capillary pressure (P_GC), distinct mechanisms may be involved in this response.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Original tracing illustrating effects of pressure (solid line) increase on afferent arteriole diameter (circles and dashed line) in isolated perfused hydronephrotic kidney. Note that elevated pressure elicits afferent arteriolar vasoconstriction without a preceding stretch (no initial increase in diameter). Diameter and pressure measurements were acquired at a sampling rate of 3 Hz.

We used the in vitro perfused hydronephrotic rat kidney model to directly examine the effects of modulating PKC activity on afferent arteriolar myogenic reactivity (i.e., sensitivity to elevated pressure). The operating range and response frequency of myogenic vasoconstriction in this preparation are identical to those observed for renal autoregulation in vivo (4, 21). The effects of pharmacological interventions and pathophysiological processes on myogenic reactivity in the model also correspond closely to effects on renal autoregulation in the intact kidney (6-8, 21, 22). These considerations and the lack of TGF make this model an ideal preparation for investigations of the afferent arteriolar myogenic response.

Our results indicate that PKC activity is an important modulator of myogenic afferent arteriolar vasoconstriction and that reactivity to pressure is attenuated or potentiated by manipulations that inhibit or activate PKC, respectively. Indirect evidence suggests that the influence of basal PKC on myogenic reactivity involves modulation of 4-aminopyridine-sensitive voltage-gated K channels. Finally, our results suggest that although basal PKC activity is an important determinant of afferent arteriolar myogenic reactivity, activation of PKC is not a requisite component of the signal transduction pathway mediating myogenic vasoconstriction in this vessel, as full myogenic activity can be restored in the presence of PKC inhibition.

	METHODS

Top Abstract Introduction Methods Results Discussion References

The in vitro perfused hydronephrotic rat kidney preparation was used to assess myogenic reactivity of renal afferent arterioles. Unilateral hydronephrosis was induced by ligation of the left ureter in male Sprague-Dawley rats (150 g) under halothane-induced anesthesia. Within 6-8 wk, the tubules of the hydronephrotic kidney underwent complete atrophy, allowing direct visualization of the renal microcirculation. At this stage, the rats were anesthetized (methoxyflurane) and the renal artery of the hydronephrotic kidney was cannulated in situ. The kidney was then excised and perfused in vitro. During the in vivo cannulation and throughout the excision process, kidneys were continuously perfused to avoid any disruption of nutritive flow.

The perfusion apparatus used in the present study employed a single-pass presentation of medium to the kidney. Medium was pumped on demand through a heat exchanger to a pressurized reservoir, supplying the renal artery. Perfusion pressure was monitored at the level of the renal artery and was controlled by adjusting the pressure within the perfusion reservoir. An automated pressure-control system coupled to a computer was custom-manufactured for this purpose. Kidneys were perfused with modified Dulbecco's medium containing (in mM) 1.6 Ca, 30 bicarbonate, 5 glucose, 1 pyruvate, and 5 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). The perfusate was equilibrated with 5% CO₂-95% air (PO₂ = 150 Torr). Temperature and pH were maintained at 37°C and 7.40, respectively. In the present studies, 100 µM ibuprofen was added to eliminate the production of renal prostaglandins. All agents were added directly to the perfusate.

The hydronephrotic kidney was perfused in a heated chamber on the stage of an inverted microscope. A fiber-optic probe was used to stabilize and transilluminate a portion of the membranous renal cortex. Video images of the afferent arteriole were digitized, and diameters were determined by on-line image processing. Proximal segments of the afferent arteriole (10-20 µm in length) were scanned at 0.3- to 0.5-s intervals. Diameters were measured over the entire segment length. Mean diameter values were then obtained during the plateau of the response. Typically, each final value was derived from the mean of 30-60 individual measurements, each in turn representing the mean diameter over the length of the arteriolar segment (method described in detail in Ref. 21).

Preparations were allowed to equilibrate for at least 1 h before experimental manipulations were initiated. Myogenic reactivity was assessed by monitoring afferent arteriolar diameters as renal arterial pressure was first reduced from a basal perfusion pressure of 80 mmHg to 40 mmHg and then elevated to 180 mmHg in 20-mmHg steps. Pressures were held for 1 min at each level. The afferent arteriole responds rapidly to pressure steps of this magnitude, reaching steady state within 10 s (21). Reproducible myogenic responses can be elicited for several hours in this preparation. In each experimental protocol, replicate control pressure ramps were first obtained. Kidneys were then treated with phorbol ester, chelerythrine, ANG II, or endothelin-1 for at least 15 min, and replicate pressure ramps were repeated. Replicate values for afferent arteriolar diameter at each pressure were averaged for control and experimental periods.

ANG II, endothelin-1, and 4-aminopyridine were obtained from Sigma Chemical (St. Louis, MO). Phorbol 12-myristate 13-acetate (PMA), 4-PMA, chelerythrine chloride, and ibuprofen were obtained from Research Biochemicals International (Natick, MA). All other reagents were obtained from GIBCO (Grand Island, NY).

Data were expressed as means ± SE (as an index of dispersion). The number of replicates (n) refers to the number of kidneys or vessels in each group, as only one vessel was studied in each kidney. Differences between means were evaluated by analysis of variance followed by Student's t-test (paired or unpaired). P < 0.05 was considered significant. The Bonferroni correction was utilized for multiple comparisons. In such cases, P values < 0.05/n (where n = number of comparisons) were considered significant.

	RESULTS

Top Abstract Introduction Methods Results Discussion References

Effects of modulators of PKC on afferent arteriolar myogenic reactivity. To determine if modest increases in PKC activation altered myogenic reactivity, we examined the effects of 1.0 and 10 nM PMA on pressure-induced afferent arteriolar vasoconstriction (Fig. 2). Under control conditions, elevating renal perfusion pressure elicited a graded pressure-dependent afferent arteriolar vasoconstriction at renal arterial pressures of 100-180 mmHg. The lower concentration of PMA (1.0 nM) had no effect on this response (P > 0.5, n = 5). At 10 nM, PMA did not alter afferent arteriolar diameter at low pressure (control 19.8 ± 0.6 µm vs. PMA 19.9 ± 0.6 µm at 40 mmHg; P > 0.20, n = 8) but markedly potentiated the response of the afferent arteriole to elevated pressure. After PMA treatment, the threshold for pressure-induced vasoconstriction was reduced and the magnitude of the vasoconstrictor response was enhanced at all pressures >80 mmHg (Fig. 2, P < 0.05, n = 8). Additional experiments were conducted using the inactive phorbol ester 4-PMA. This analog shares physicochemical characteristics with PMA but does not activate PKC and, as depicted in Fig. 3, had no effect on myogenic reactivity (P > 0.50 for all data points, n = 4).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Potentiation of myogenic reactivity by phorbol 12-myristate 13-acetate (PMA). A: original tracing. B: mean data. PMA at 10 nM had no effect on basal afferent arteriolar diameter but markedly potentiated pressure-induced vasoconstriction. * P < 0.05 vs. control (n = 8).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Unlike PMA (see Fig. 2), the inactive phorbol analog 4-PMA had no effect on myogenic reactivity. A: original tracing. B: mean data. , 4-PMA; , control data. P > 0.50 at all pressures (n = 4).

To further examine the role of basal PKC activity on myogenic reactivity, we determined the effects of the PKC inhibitor chelerythrine. Chelerythrine caused a dose-dependent inhibition of myogenic reactivity, significantly reducing responsiveness at 1.0 µM (Fig. 4). However, 1.0 µM chelerythrine was found to be sufficient to completely prevent the potentiating actions of 10 nM PMA (Fig. 5). Furthermore, whereas 1.0 µM chelerythrine prevented the actions of PMA, indicating inhibition of PKC, this concentration had no effect on KCl-induced vasoconstriction (Fig. 6). Thus 35 mM KCl reduced afferent arteriolar diameters from 13.5 ± 0.5 to 3.7 ± 0.6 µm in controls and from 13.1 ± 0.5 to 3.6 ± 0.3 µm after 1.0 µM chelerythrine treatment (P = 0.38, n = 8). Increasing the concentration of chelerythrine to 3.0 µM resulted in a significant inhibition of KCl-induced afferent arteriolar vasoconstriction (35 mM KCl reduced diameters from 14.1 ± 0.4 to 4.0 ± 0.9 µm in controls and from 15.4 ± 0.7 to 8.1 ± 1.5 µm after treatment with 3.0 µM chelerythrine; P = 0.017, n = 5). Because these findings indicated that 1.0 µM chelerythrine was effective in completely preventing PKC activation by PMA, but that higher concentrations might attenuate afferent arteriolar reactivity by nonselective actions, 1.0 µM chelerythrine was used to inhibit PKC in all subsequent experiments.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Inhibition of protein kinase C (PKC) by chelerythrine attenuated basal myogenic reactivity at 1.0 µM (n = 10) and 10 µM (n = 4) chelerythrine. A: original tracing. B: mean data. * P < 0.05 vs. control.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Pretreatment with 1.0 µM chelerythrine completely prevented the potentiating effects of 10 nM PMA. A: original tracing. B: mean data (n = 7).

View larger version (24K): [in this window] [in a new window]   Fig. 6.   At 1.0 µM, chelerythrine did not alter afferent arteriolar response to KCl-induced depolarization. Open bars, basal diameters; filled bars, diameters after treatment with 35 mm KCl. Note that chelerythrine did not alter basal diameters or response to KCl (n = 8, P = 0.38).

Effects of ANG II and endothelin on myogenic reactivity. PKC has been implicated in the afferent arteriolar actions of both ANG II and endothelin. To determine if physiological means of altering basal PKC activity also modulate myogenic reactivity, we examined the actions of low concentrations of these agents. Preliminary experiments were conducted to establish the appropriate concentration range. At 30 and 100 pM, ANG II constricted the afferent arteriole by 23 ± 6 and 44 ± 4%, respectively (n = 5), when perfusion pressure was maintained constant at 80 mmHg. Under these conditions 3 pM ANG II did not elicit a vasoconstrictor response (diameters: control, 17.4 ± 0.8 µm; 3 pM ANG II, 17.3 ± 0.8 µm; P > 0.5, n = 12). As depicted in Fig. 7, however, this low concentration of ANG II significantly potentiated myogenic reactivity. Thus, in the presence of 3 pM ANG II, the threshold for pressure-induced vasoconstriction was shifted to the left, and the magnitude of the vasoconstriction (at pressures >100 mmHg) was significantly enhanced. These actions of ANG II were similar to that observed for PMA (Fig. 2). We next determined whether the effects of ANG II were dependent on PKC, using the same conditions shown to block the actions of PMA (1.0 µM chelerythrine). As depicted in Fig. 8, in the presence of chelerythrine, 3 or 10 pM ANG II had no effect on the myogenic response, indicating that 3 pM ANG II potentiates myogenic reactivity by activating PKC. As a positive control, we found that the L-type Ca channel agonist BAY K 8644 was able to potentiate myogenic vasoconstriction in this setting (data not shown), indicating that myogenic responses can be influenced by PKC-independent mechanisms in the presence of chelerythrine.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   ANG II-induced potentiation of myogenic reactivity. A: original tracings illustrate afferent arteriolar response to elevated renal perfusion pressure under control conditions (left) and after treatment with 3 pM ANG II (right). B: mean data illustrating that ANG II treatment did not alter basal arteriolar diameter but potentiated pressure-induced vasoconstriction (n = 12, * P < 0.05 vs. control).

View larger version (28K): [in this window] [in a new window]   Fig. 8.   Chelerythrine (1.0 µM) completely prevented ANG II-induced potentiation of myogenic reactivity. A: original tracing. B: mean data demonstrate lack of effect of 3 pM (shaded squares) or 10 pM (solid squares) ANG II on myogenic response after 1.0 µM chelerythrine (open squares) (n = 4).

View larger version (21K): [in this window] [in a new window]   Fig. 9.   Endothelin-1 (ET-1)-induced potentiation of myogenic reactivity. Like ANG II (see Fig. 7), 3 pM ET-1 markedly enhanced myogenic vasoconstriction. At this low concentration, ET-1 had no effect on basal diameter. A: original tracing. B: mean data (n = 6, * P < 0.05 vs. control).

View larger version (29K): [in this window] [in a new window]   Fig. 10.   Inhibition of PKC prevented ET-1-induced potentiation of myogenic reactivity. A: original tracing illustrating protocol. B: mean data illustrating effects of ET-1 on myogenic responses after pretreatment with 1.0 µM chelerythrine. Chelerythrine prevented ET-1-induced potentiation (3 pM and 10 pM ET-1) and unmasked an ET-1-induced inhibition of myogenic vasoconstriction [* P < 0.05 vs. chelerythrine alone () (n = 5)].

View larger version (35K): [in this window] [in a new window]   Fig. 11.   4-Aminopyridine (4-AP) reversed the actions of chelerythrine. Treatment with 1.0 µM chelerythrine () attenuated basal myogenic response (* P < 0.05 vs. control). Subsequent treatment with 1 mM 4-AP fully restored myogenic reactivity to control levels (n = 8).

View larger version (32K): [in this window] [in a new window]   Fig. 12.   Pretreatment with 4-AP prevented the actions of chelerythrine. 4-AP (1.0 mM) caused a modest, albeit significant, potentiation of myogenic reactivity (* P < 0.05 vs. control; n = 8). Subsequent treatment with chelerythrine did not result in attenuation of myogenic reactivity (as seen in controls; e.g., see Fig. 11).

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

The present study is the first to examine the role of PKC in the myogenic vasoconstrictor response of the renal afferent arteriole. Our results indicate that PKC activity is an important determinant of myogenic reactivity in this vessel, in that PKC inhibition attenuated and PKC stimulation augmented pressure-induced vasoconstriction. We suggest that the activation of PKC is not a requisite event in afferent arteriolar myogenic signaling but rather that basal PKC activity plays a permissive role by modulating the basal activity of voltage-activated K channels, thereby indirectly influencing pressure-induced depolarization.

In the present study, chelerythrine attenuated basal myogenic reactivity at a concentration of 1 µM. At this concentration chelerythrine selectively inhibits PKC in vitro (9) and completely prevented the potentiating effects of 10 nM PMA in our study, indicating an effective blockade of PKC. This concentration of chelerythrine also attenuated myogenic vasoconstriction but had no effect on the vasoconstrictor response to KCl-induced depolarization. Higher concentrations of chelerythrine further attenuated myogenic reactivity but also affected KCl-induced vasoconstriction, suggesting a nonspecific action. These data demonstrate that 1 µM chelerythrine is an effective means of manipulating PKC in our preparation and that blockade of PKC produces an inhibition of basal afferent arteriolar myogenic reactivity.

There are two possible interpretations of this finding. One is that PKC plays an obligate role in myogenic signaling and that blockade of this pathway thereby attenuates myogenic signal transduction. In support of this interpretation, pressure and stretch have been shown to increase phospholipase C activity and elevate diacylglycerol and D-myo-inositol 1,4,5-trisphosphate levels in vascular myocytes (15, 29). Furthermore, it has been suggested that a stimulation of PKC is required for myogenic vasoconstriction (10, 16, 25, 30, 31). However, another possible interpretation of this finding is that myogenic vasoconstriction of the afferent arteriole is not necessarily dependent on a pressure-induced stimulation of PKC but rather that myogenic reactivity is sensitive to the basal level of PKC activity. In other words, the underlying level of PKC activity plays a permissive role in the myogenic response by interacting synergistically with myogenic mechanisms. Although the actions of chelerythrine could be explained by either of the above interpretations, we believe that, in concert, our findings predominantly support the latter hypothesis.

The ability of low levels of PMA, ANG II and endothelin-1 to potentiate pressure-induced vasoconstriction lends support to this postulate. At the concentration employed (3 pM), ANG II and endothelin-1 did not alter basal afferent arteriolar diameter but augmented myogenic reactivity in a PKC-dependent manner. A reasonable interpretation of these observations is that low levels of receptor- or PMA-induced PKC activation interact synergistically with myogenic vasoconstrictor mechanisms. If activation of PKC were a requisite component of both myogenic and receptor-mediated signaling, one would anticipate a convergence of the two signaling pathways. The observed synergism indicates a divergence in the pathways activated by elevated pressure and receptor activation and suggests that the latter involves activation of PKC. Admittedly a caveat of this argument is that agonists and pressure may activate PKC by independent mechanisms and produce additive effects on PKC activity but synergistic effects on contractility.

More compelling evidence against an obligate role of PKC activation in myogenic signaling is the observation that 4-aminopyridine restored normal myogenic reactivity in the continued presence of PKC inhibition. As discussed below, we hypothesize that basal PKC modulates myogenic reactivity by exerting a tonic inhibitory effect on voltage-activated K channels. Pharmacological inhibition of these K channels by 1 mM 4-aminopyridine appeared to circumvent the effects of PKC inhibition and to fully restore normal myogenic reactivity (Fig. 11). Furthermore, pretreatment with 4-aminopyridine prevented the inhibitory effects of subsequent administration of chelerythrine. The ability of the afferent arteriole to exhibit normal myogenic vasoconstriction in the presence of PKC inhibition in these experiments argues against a direct and obligate role of PKC activation in myogenic signaling of this vessel.

It should be noted that potentiation of stretch- and pressure-induced vasoconstriction by PKC is observed in other vessel types (17, 18, 30). Vasoconstrictor agonists have also been shown to potentiate myogenic responses by PKC-dependent mechanisms in other preparations (19, 26). Similarly, elevated pressure and stretch enhance the reactivity of isolated vessel segments to vasoconstrictor agonists (20, 27, 32), presumably by the same mechanism. Of interest, Yuan et al. (35) found that elevated pressure did not potentiate responsiveness to ANG II in the isolated renal afferent arteriole, a finding in direct contradiction to the present observations. However, the afferent arteriolar preparation employed in the study by Yuan et al. (35) lacked myogenic reactivity. Thus failure of transmural pressure to influence agonist-induced activation in this setting may simply reflect a general impairment of the myogenic mechanism in this preparation.

There are a number of possible mechanisms whereby PKC might interact synergistically with myogenic activation. Pressure-induced afferent arteriolar vasoconstriction is abolished by Ca antagonists (6) and is sensitive to K-channel-induced hyperpolarization (24), indicating a dependence on depolarization-induced Ca entry. In contrast, phorbol esters elicit contractile responses that are independent of altered Ca influx or increased cytosolic [Ca] (12, 13), suggesting altered Ca sensitivity. PKC is known to phosphorylate the actin-binding protein calponin, thereby removing calponin-induced inhibition of actin-myosin ATPase and modulating contractility without altering Ca signaling or myosin light-chain phosphorylation (34). It is relevant in this regard that PMA potentiates stretch-induced vasoconstriction of the rabbit facial vein without affecting Ca influx, intracellular Ca, or myosin light-chain phosphorylation (18).

PKC has also been shown to regulate the activity of voltage-gated K channels, and it is this mechanism that we believe more closely agrees with our observations. Both ANG II (2) and phorbol ester (1) have been shown to inhibit K_dr through activation of PKC. The open probability of K_dr is increased by membrane depolarization, and the activation of this channel may normally buffer the effects of membrane-depolarizing stimuli (3, 14). Thus inhibition of this voltage-activated outward current by the basal activity of PKC may facilitate pressure-induced depolarization and thereby play a permissive role in myogenic vasoconstriction. In support of this postulate, we found that 4-aminopyridine, an inhibitor of K_dr, reversed the effects of chelerythrine, as would be anticipated if this treatment mimicked the effects of (basal) PKC. Similarly, pretreatment with 4-aminopyridine caused a modest potentiation of myogenic vasoconstriction and prevented any depressor effects of subsequent PKC inhibition. In concert, these findings support our postulate that inhibition of basal PKC modulates myogenic reactivity by removing a tonic inhibition of 4-aminopyridine-sensitive K channels. The fact that PKC inhibition attenuated myogenic reactivity but did not affect vasoconstrictor response to KCl (Fig. 6) is consistent with this interpretation. Elevated K shifts the K equilibrium potential and eliminates the effects of altered K conductance on membrane potential and reactivity, and we have previously demonstrated that KCl-induced afferent arteriolar vasoconstriction is insensitive to altered K channel activity (24, 33). Obviously, studies examining the effects of PKC on pressure-induced afferent arteriolar depolarization will be required to fully address this hypothesis.

Finally, it is of interest that, in the presence of chelerythrine, endothelin-1 actually produced a significant and dose-dependent inhibition of myogenic vasoconstriction. Exogenous endothelin-1 activates both endothelin-A (ET_A) and endothelin-B (ET_B) receptor subtypes. Both receptor subtypes have been linked to vasoconstriction. In the kidney, evidence suggests the presence of at least two ET_B receptor subtypes, one of which appears to be linked to NO- and cyclooxygenase-dependent renal vasodilation (5, 36). A plausible explanation for our observation that, in the presence of chelerythrine, endothelin-1 exerted a vasodepressor action is that the inhibition of PKC may have unmasked a vasodilatory response mediated by endothelin-1 activation of an ET_B receptor.

In conclusion, we found myogenic reactivity to either be attenuated or potentiated by acute manipulations of PKC activity and that low levels of the endogenous renal vasoconstrictors ANG II and endothelin-1 enhance myogenic reactivity through this mechanism. We interpret these observations as indicating that basal PKC activity is an important determinant of myogenic reactivity in the renal afferent arteriole. However, pressure-induced PKC activation does not appear to play an obligate role in myogenic signaling in this vessel, as PKC blockade has no effect on myogenic reactivity in the presence of 4-aminopyridine. We suggest that the basal state of PKC activation directly modulates voltage-gated K channel activity and thereby indirectly influences myogenic reactivity.