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REGULAR ARTICLES |
*
Department of Physiology and Biophysics, University of Nebraska College of
Medicine, Omaha, Nebraska
Department of Internal Medicine, Otawara Redcross Hospital, Otawara,
Japan.
Correspondence to Pamela K. Carmines, Department of Physiology and Biophysics, University of Nebraska College of Medicine, 984575 Nebraska Medical Center,Omaha NE 68198-4575. Phone : 402-559-9353 ; Fax : 402-559-4438 ; E-mail : pkcarmin{at}unmc.edu
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Introduction |
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Although numerous metabolic and hormonal abnormalities have been implicated in the pathogenesis of diabetic hyperfiltration, no single factor has been proven to be responsible for the early changes in renal vascular function. It seems unlikely that changes in any specific membrane receptor or vasoactive agonist would underlie an altered responsiveness to a broad spectrum of stimuli, the situation that appears evident in the early stage of IDDM (4, 5). Bank and coworkers (6) have proposed that insulinopenia in IDDM might impair Ca2+ movement through renal vascular voltage-gated Ca2+ channels. Indeed, recent studies from our laboratory revealed that afferent arteriolar contractile responses to BAY K8644 (a dihydropyridine agonist) and K+-induced membrane depolarization are impaired during the hyperfiltration stage of IDDM, and that these changes are accompanied by suppressed afferent arteriolar [Ca2+] responses to membrane depolarization (7). Because voltage-gated Ca2+ channels are prominent in afferent arterioles, but functionally silent in efferent arterioles (8), altered expression or regulation of these channels would promote afferent arteriolar dilation and hyperfiltration in IDDM ; however, dysregulation of membrane potential as a result of increased K+ conductance might also contribute to this phenomenon.
Among the K+ channels that have been identified in the cell membrane of arterial smooth muscle cells (9), K+ channels closed by intracellular ATP (KATP channels) are unique in that their activity may reflect the cellular metabolic state. KATP channels are composed of pore-forming (inwardly rectifying K+ channel ; KIR 6.x) and regulatory (sulfonylurea receptor ; SUR) subunits. Distinct combinations of KIR 6.x and SUR subunits associate in a 1 : 1 stoichiometry to form tetrameric channels in specific cell types (10). The ATP-sensing mechanism and biophysical properties of KATP channels are intrinsic to the KIR 6.x subunit, while the various SUR isoforms confer tissue-specific pharmacologic properties regarding interactions with sulfonylurea compounds and potassium channel openers (11). Previous studies have provided functional evidence that KATP channels are expressed in the renal microcirculation (12,13,14,15), although the KATP 6.x and SUR subunits comprising these channels have not been identified. Marcus and coworkers (16) have proposed that changes in KATP channel activity could engender glomerular hyperfiltration in IDDM. Although numerous pharmacologic studies have assessed KATP channel function in diverse vascular beds at various times during the progression of IDDM (17,18,19,20,21), the results have been inconsistent and the impact of KATP channels on renal afferent arteriolar function during the hyperfiltration stage of IDDM remains unexplored. Accordingly, the goal of the present study was to examine the hypothesis that the influence of KATP channels on afferent arteriolar tone is exaggerated during the early (hyperfiltration) stage of IDDM in the rat.
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Materials and Methods |
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Induction of Diabetes Mellitus
Rats were anesthetized with sodium methohexital (50 mg/kg,
intraperitoneally) to facilitate intravenous injection of streptozocin (STZ)
(65 mg/kg ; Upjohn Co., Kalamazoo, MI). Sham rats received vehicle. The rats
were allowed to recover from anesthesia and were housed overnight. The next
morning, blood glucose levels were measured (Accu-Check III model 766 ;
Boehringer Mannheim, Indianapolis, IN), and the rats were anesthetized again
to allow either : (1) intraperitoneal implantation of an osmotic
minipump (model 2002 ; Alzet, Palo Alto, CA) prepared for delivery of insulin
(3.5 U/kg per d Iletin II ; Eli Lily, Indianapolis, IN) or (2)
subcutaneous insertion via a 16-gauge needle of a 2.3 x 2.0 mm
sustained-release insulin implant (Linplant® ; Linshin Canada,
Scarborough, Ontario, Canada). In our hands, both modes of insulin delivery
maintain moderate hyperglycemia during the ensuing 2-wk period. Sham rats
received either diluent infusion via a minipump or a micro-recrystallized
palmitic acid (vehicle) implant. All animals subjected to minipump
implantation received penicillin G procaine (60,000 U intramuscularly)
immediately after surgery. Thereafter, blood glucose concentration and body
weight were measured twice weekly.
In Vitro Blood-Perfused Juxtamedullary Nephron Technique
Two weeks after induction of IDDM, acute experiments were performed using
the in vitro blood-perfused juxtamedullary nephron technique
(22). Most experiments used
two animals, one as a blood donor and another as a kidney donor, although a
single rat provided both tissues in some instances. After anesthetization with
sodium pentobarbital (50 mg/kg, intraperitoneally), a catheter was placed in
the carotid artery. Arterial pressure was measured in some animals via this
carotid cannula using a P23XL transducer (Gould, Oxnard, CA) connected to a
polygraph (Grass Instruments, Quincy, MA). Blood donors were bilaterally
nephrectomized through flank incisions, and their blood was collected into a
heparinized syringe. Kidney donors were laparotomized and the left kidney was
removed. A perfusion cannula introduced via the superior mesenteric artery
into the right renal artery allowed renal perfusion with Tyrode's solution
containing 52 g/L bovine serum albumin, a mixture of L-amino acids
(23), and either 5 or 20 mM
glucose (in sham and STZ rats, respectively). The renal vein was incised to
drain the perfusate and the animal was exsanguinated into a heparinized
syringe ; however, renal perfusion was maintained throughout the ensuing
dissection procedure. The kidney was removed and cut longitudinally to expose
the pelvic cavity, leaving the papilla intact within the perfused dorsal
two-thirds of the organ. Small incisions were made in the lateral fornices,
allowing the papilla to be reflected back and retained in that position by
insect pins. The pelvic mucosa, adipose and connective tissues that normally
cover the inside cortical surface were removed and the veins were cut open,
thus exposing the tubules and microvasculature of juxtamedullary nephrons. The
portions of the vasculature that give rise to arterioles associated with these
juxtamedullary nephrons were isolated with tight ligatures.
Blood collected from the two rats was prepared for perfusion as described previously (7). The reconstituted blood was filtered through a 5-µm nylon mesh, and its pH was measured (System 1302 pH/Blood Gas Analyzer ; Instrumentation Laboratory, Lexington, MA) and adjusted to 7.40 to 7.44 by addition of NaHCO3, as necessary. Thereafter, the blood was stirred continuously in a closed reservoir that was maintained under pressure from a gas tank (95% O2/5% CO2). This arrangement provided both tissue oxygenation and the driving force for perfusion of the kidney. The Tyrode's perfusate was then replaced by the reconstituted blood perfusate. Kidneys from STZ rats were perfused with blood from STZ donors, while kidneys from sham rats were perfused with blood from sham donors. Perfusion pressure measured at the cannula tip in the renal artery was maintained at 110 mmHg throughout the experiment. The tissue was warmed to 37°C, and its surface was continuously bathed with Tyrode's solution containing 10 g/L bovine serum albumin, approximating the composition of renal interstitial fluid (24). Stock solutions containing vasoactive agents (100 mM glibenclamide in DMSO ; 150 mM pinacidil in EtOH ; 10 mM PCO-400 in DMSO) were stored at -20°C until the day of the experiment, at which time they were diluted with Tyrode's bath to the appropriate final concentrations.
Videometric techniques were used to measure arteriolar lumen diameter (7). The tissue was transilluminated on the fixed stage of a Nikon Optiphot microscope equipped with a water-immersion objective (x40, numerical aperture 0.55). Enhanced video images of the microvessels were displayed at a magnification of x1400 on a high-resolution monitor (Conrac Display Systems, Covina, CA) and recorded simultaneously on videotape using a SuperVHS videocassette recorder (Panasonic, Secaucus, NJ). During the 15-min equilibration period, an afferent arteriole was selected for study based on visibility and acceptable blood flow. Only arterioles with rapid flow of erythrocytes were studied, and vessels were rejected on the basis of inadequate flow if the passage of single erythrocytes could be discerned. All experimental protocols were designed to assess arteriolar diameter at a single measurement site under several experimental conditions. When two vessels could be visualized within the same field of view, responses of both vessels were recorded simultaneously and analyzed separately during videotape playback. Afferent arteriolar lumen diameter was monitored at sites located >100 µm upstream from the glomerulus and >80 µm downstream from the interlobular artery.
Experimental Protocols
Afferent Arteriolar Diameter Responses to Glibenclamide.
The effect of glibenclamide (Sigma Chemical, St. Louis, MO), a potent and
selective KATP channel inhibitor
(10), on afferent arteriolar
tone was examined in kidneys from STZ and sham rats. Afferent arteriolar
diameter responses to this drug were assessed by sequential exposure of the
tissue surface to the following solutions : (1) Tyrode's bath alone,
10 min ; (2) Tyrode's solution containing 3, 10, 30, 100, and 300
µM glibenclamide for 5 min at each concentration ; and (3)
Tyrode's solution alone, 15 min. Previous studies from our laboratory have
documented the stability of perfused juxtamedullary afferent arteriolar
diameter in normal rat kidney for at least 35 min
(25,26).
To exclude the possibility that renal arterioles from STZ rats are more
susceptible to time-related degeneration than arterioles from normal kidneys,
a time-control study was performed on kidneys from six STZ rats. In this
time-control study, afferent arteriolar lumen diameter was monitored while the
tissue surface was exposed to Tyrode's solution alone for 50 min.
Impact of Baseline Diameter on Afferent Arteriolar Responses to
Glibenclamide.
Additional experiments were performed to determine whether a nonspecific
effect of increased arteriolar baseline diameter per se could
underlie the exaggerated response to glibenclamide observed in kidneys from
STZ rats. Preliminary experiments revealed that treatment of kidneys from sham
rats with 5 µM sodium nitroprusside (SNP) (Elkins-Sinn, Cherry Hill, NJ)
evoked prompt and sustained afferent arteriolar dilation, achieving lumen
diameters similar to those typically observed under baseline conditions in
kidneys from STZ-treated rats. Accordingly, juxtamedullary afferent arterioles
in kidneys from sham rats were exposed sequentially to the following bathing
solutions : (1) Tyrode's solution alone, 10 min ; (2)
Tyrode's solution containing 5 µM SNP, 5 min ; (3) Tyrode's
solution containing both 5 µM SNP and increasing concentrations (3 to 300
µM) of glibenclamide, 5 min at each concentration ; (4) Tyrode's
solution containing 5 µM SNP, 15 min ; and (5) Tyrode's solution
alone.
Afferent Arteriolar Diameter Responses to Pinacidil.
Afferent arteriolar responses to the cyanoguanidine
KATP agonist pinacidil
(27,
28) were assessed in kidneys
from STZ and sham rats. The protocol used sequential exposure of the tissue
surface to the following bathing solutions : (1) Tyrode's solution
alone, 10 min ; (2) Tyrode's solution containing 0.3, 1.0, 3.0, 10,
30, 100, and 300 µM pinacidil for 5 min at each concentration ; and
(3) Tyrode's solution alone, 15 min. The duration of exposure to the
drug was determined by preliminary experiments conducted using kidneys from
normal rats (n = 3), in which the dilator response of afferent
arterioles to 300 µM pinacidil developed almost immediately, reached its
maximum within 3 min, and was sustained for more than 40 min.
Glibenclamide Reversal of Pinacidil-Induced Vasodilation.
Additional experiments were performed to confirm that both pinacidil and
glibenclamide influence afferent arteriolar function via actions on the same
effector, presumably KATP channels. Afferent arterioles
from normal rats (n = 5) were exposed to the following bathing
solutions : (1) Tyrode's solution alone, 10 min ; (2)
Tyrode's solution containing 30 µM pinacidil, 5 min ; (3) Tyrode's
solution containing both 30 µM pinacidil and 100 µM glibenclamide, 15
min ; and (4) Tyrode's solution containing 30 µM pinacidil alone,
5 min.
Arteriolar Responses to PCO-400 in the Absence and Presence of
Glibenclamide.
A final set of experiments assessed the effect of IDDM on afferent
arteriolar dilator responses to PCO-400 (BIOMOL, Plymouth Meeting, PA), a
benzopyran KATP antagonist
(29). Afferent arteriolar
responses to 1 µM PCO-400 (a concentration selected based on preliminary
experiments using kidneys from normal rats) were monitored in kidneys from
sham and STZ rats before, during, and after exposure to 100 µM
glibenclamide.
Statistical Analyses
Microvessel lumen diameter was determined from videotaped images using a
digital image-shearing monitor (model 908 ; IPM, San Diego, CA). This device
was calibrated using a stage micrometer (smallest division = 2 µm) and
yielded diameter measurements reproducible to within <1 µm. Lumen
diameter was measured at 12-s intervals from a single site along the vessel
length. The average diameter during the final minute of each treatment period
was used for statistical analysis of responses. Data were analyzed by ANOVA
for repeated measures or Friedman repeated-measures ANOVA on ranks, as
appropriate, followed by Newman-Keuls test. A P value<0.05 was
considered statistically significant. All data are presented as the mean
± SEM (n = number of arterioles, unless otherwise stated).
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Afferent arteriolar function was assessed at sites 350 ± 30 µm from the glomerulus in STZ-treated rats and 210 ± 30 µm from the glomerulus in sham rats (P <0.05). However, this difference loses its significance when corrected by total length of afferent arterioles, and measurement was conducted at 42 ± 17 and 43 ± 17% of the total length from the glomerulus in each group of rats. In accord with our previous observations (30, 31), STZ-treated rats had significantly larger baseline afferent arteriolar diameter than sham/normal rats, with values averaging 25.6 ± 0.9 (n = 28) and 20.5 ± 0.8 µm (n = 26), respectively.
Afferent Arteriolar Diameter Responses to Glibenclamide
The basal impact of KATP channels on afferent
arteriolar diameter in kidneys from sham and STZ rats was evaluated based on
responses to increasing concentrations of glibenclamide in both groups of
rats. Although kidneys from sham rats exhibited a modest but statistically
significant decrease in afferent arteriolar diameter when exposed to the
highest concentration of glibenclamide (300 µM ; 
= -1.7 ±
0.9 µm ; n = 8 ; P < 0.05), the drug had no effect on
these vessels at lower concentrations. In contrast, glibenclamide evoked
concentration-dependent reductions in afferent arteriolar lumen diameter in
kidneys from STZ rats, with significant changes evident in response to 30
µM glibenclamide ( = -2.4 ± 0.6 µm ; n = 7 ;
P < 0.05). During exposure of STZ kidneys to 300 µM
glibenclamide, afferent diameter was 5.5 ± 1.1 µm lower than the STZ
baseline and similar to baseline values observed in sham kidneys. Upon removal
of glibenclamide from the bath, afferent arteriolar diameter was restored to
19.1 ± 1.6 µm in sham kidneys and 25.0 ± 2.6 µm in STZ
kidneys (P > 0.05 versus baseline values). Time-control
experiments revealed no significant change in afferent arteriolar diameter in
kidneys from STZ rats within the time frame of these experiments (baseline
diameter = 26.2 ± 2.6 µm ; diameter after 50 min exposure to
Tyrode's bathing solution = 27.0 ± 2.8 µm ; n = 7). Thus,
although glibenclamide had little impact on afferent arterioles in kidneys
from sham rats, the observation of significant afferent constrictor responses
to glibenclamide in kidneys from STZ rats suggests that open
KATP channels exert a basal dilator influence on the
afferent arteriole in IDDM.
Impact of Baseline Diameter on Responses to Glibenclamide
Figure 1 also shows the
effect of glibenclamide on predilated afferent arterioles in kidneys from sham
rats. In these experiments, exposure to 5 µM SNP increased arteriolar
diameter from 21.3 ± 2.6 to 28.9 ± 1.5 µm (n = 4), a
value that does not differ significantly from baseline afferent diameter in
kidneys from STZ rats. During continued exposure to SNP, increasing
concentrations of glibenclamide had no effect on these predilated vessels
( = 1.6 ± 0.9 µm during treatment with 300 µM
glibenclamide ; not significant versus SNP alone). Removal of
glibenclamide from the SNP-containing bath did not alter afferent diameter
(31.2 ± 1.9 µm), while subsequent removal SNP from the bath restored
diameter to a value similar to baseline (22.3 ± 2.4 µm). Thus, the
afferent arteriolar constrictor response to glibenclamide in kidneys from STZ
rats cannot be attributed to a nonspecific effect of reduced basal arteriolar
tone per se.
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Afferent Arteriolar Diameter Responses to Pinacidil
Additional experiments were performed to determine whether the potentiated
afferent arteriolar response to glibenclamide in STZ kidneys reflects opening
of recruitable KATP channels that are normally present
but quiescent under basal conditions. This issue was evaluated on the basis of
afferent arteriolar diameter responses to increasing concentrations of
pinacidil (Figure 2). In
kidneys from STZ-treated rats, 1 µM pinacidil evoked a significant increase
in afferent arteriolar diameter ( = 4.1 ± 1.2 µm ; n
= 8), whereas this concentration of pinacidil had no effect on afferent
diameter in kidneys from sham rats = 0.0 ± 0.4 µm ; n = 9).
Moreover, responses to 300 µM pinacidil were significantly greater in STZ
rats ( = 18.1 ± 2.1 µm) than in sham rats ( = 10.1
± 1.2 µm). In a subset of four kidneys from sham rats, subsequent
exposure to 1 mM pinacidil did not evoke any further increase in arteriolar
diameter, indicating that the maximum response to this agonist had been
achieved. Thus, while pinacidil evoked a concentration-dependent increase in
afferent arteriolar lumen diameter in kidneys from both groups of rats,
vessels from STZ-treated rats were more sensitive to the drug and the
magnitude of the response was augmented substantially compared to vessels from
sham rats. Removal of pinacidil from the superfusate bath restored afferent
arteriolar diameter to 22.3 ± 1.3 µm in sham kidneys and 25.1
± 2.4 µm in STZ kidneys (both P > 0.05 versus
baseline), indicating that the observed responses did not reflect a
time-related loss of arteriolar tone. The observation of exaggerated dilator
responses to pinacidil in afferent arterioles from STZ kidneys suggests that
the functional availability of KATP channels may be
increased in IDDM.
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Glibenclamide Reversal of Pinacidil-Induced Vasodilation
Figure 3 documents the
ability of glibenclamide to reverse the afferent arteriolar dilator response
to pinacidil in kidneys from normal rats. Exposure to 30 µM pinacidil
increased afferent arteriolar lumen diameter from 17.5 ± 0.9 to 24.5
± 1.6 µm (P < 0.05 ; n = 5). Subsequent
addition of 100 µM glibenclamide to the pinacidil-containing bath
significantly reduced arteriolar diameter to 21.6 ± 1.8 µm within 5
min. After 15 min of simultaneous exposure to both drugs, arteriolar diameter
averaged 18.2 ± 0.8 µm (not significant versus initial
baseline). When glibenclamide was removed from the pinacidil-containing bath,
the vessels dilated again to 23.6 ± 1.6 µm (P < 0.05
versus baseline value ; not significant versus initial
response to pinacidil alone).
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Effect of IDDM on Responses to PCO-400
The ability of KATP agonists to elicit exaggerated,
sulfonylurea-sensitive afferent arteriolar vasodilation in IDDM was confirmed
in studies using PCO-400, a KATP opener that is
structurally distinct from pinacidil
(28). In these experiments,
baseline afferent arteriolar diameter averaged 19.3 ± 0.5 µm
(n = 5) in kidneys from sham rats and 24.6 ± 1.9 µm in STZ
kidneys (n = 6 ; P < 0.05 versus sham). In each
arteriole studied, diameter responses to PCO-400 were monitored before,
during, and after glibenclamide treatment. PCO-400 responses obtained after
removal from glibenclamide from the bath did not differ significantly from
those evident before glibenclamide treatment. Therefore, the average response
for each vessel was used for data analysis and for the preparation of
Figure 4, which summarizes
responses to PCO-400 and the glibenclamide sensitivity of these responses in
sham and STZ kidneys. In kidneys from sham rats, 1 µM PCO-400 significantly
increased afferent arteriolar diameter by 2.6 ± 0.7 µm. Although 100
µM glibenclamide failed to alter baseline diameter in sham kidneys, it
abolished the dilator response to PCO-400 ( = 0.4 ± 0.6 µm).
In kidneys from STZ rats, 1 µM PCO-400 increased afferent arteriolar
diameter by 6.2 ± 1.3 µm (P < 0.05 versus sham
kidneys). During exposure of STZ kidneys to 100 µM glibenclamide, afferent
diameter was 3.0 ± 0.8 µm lower than the STZ baseline (P
< 0.05) and did not differ from baseline values observed in sham kidneys.
Glibenclamide exposure also abolished the vasodilator response to PCO-400 in
STZ kidneys ( = -0.2 ± 0.3 µm).
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Because of the heteromultimeric nature of KATP channels, the standard sulfonylurea blockers and the structurally heterogeneous array of potassium channel openers display a wide spectrum of affinities toward KATP channels expressed in varied tissues. Nevertheless, these well characterized pharmacologic tools have yielded substantial insight into the functional role of KATP channels in a variety of physiologic and patho-physiologic settings. The native SUR2B/KIR6.2 channel expressed in most vascular smooth muscle is inhibited by 20 µM glibenclamide (the most potent sulfonylurea, a derivative of which was exploited in the initial purification of the SUR subunit) and activated by low micromolar concentrations of pinacidil (10, 33). In the present study, significant effects of glibenclamide on afferent arteriolar diameter were observed in kidneys from STZ rats only at bath concentrations of 30 to 300 µM. Glibenclamide binds with high affinity to bovine serum albumin (present in our superfusate bath at a concentration of 10 g/L), such that only 1 to 10% of total drug is available in free form in media similar to that used in the present study (34, 35). Accordingly, the maximum free glibenclamide concentration achieved in our experiments was in the range of 3 to 30 µM, which should block vascular smooth muscle KATP channels while exerting little effect on other classes of ion channel. Available data indicate that 50 to 90% of pinacidil is available in free form in albumin-containing solutions (36). Our studies reveal that the effect of pinacidil on juxtamedullary afferent arteriolar diameter is rapid in onset and fully reversed by glibenclamide within 15 min. A similar time course for achieving maximal inhibition of the pinacidil response has been reported to occur in rabbit superior mesenteric artery (37) and was suggested to indicate a requirment for glibenclamide to enter the lipid bilayer and move laterally within the membrane to access its site of action (38). Glibenclamide and pinacidil bind to the SUR subunit of the channel at separate cytoplasmic sites, and the prolonged time course for glibenclamide reversal of pinacidil-induced dilation may reflect the fact that cyanoguanidine binding inhibits sulfonylurea binding in a negative allosteric manner (17). Overall, the pharmacologic properties of glibenclamide and pinacidil at free concentrations achieved in the present study are consistent with the well established and specific actions of these agents on vascular KATP channels. The ability of glibenclamide to restore afferent arteriolar diameter after dilation by pinacidil, and to abrogate dilator responses to PCO-400 in both sham and STZ rats, lends further credence to the contention that these agents act on a common target in our experimental setting—presumably KATP channels in afferent arteriolar smooth muscle cells.
In normal rats, systemic infusion of KATP blockers has been reported to increase (39) or not alter renal vascular resistance and GFR (40,41,42). Reductions in medullary blood flow have also been reported (43,44). The effects of these agents on renal hemodynamics in vivo can be complicated by alterations in arterial pressure or reflex-mediated responses that allow maintained arterial pressure. Overall, however, the literature indicates that KATP blockers exert little direct impact on renal vascular resistance under basal conditions. The ineffectiveness of glibenclamide as an afferent arteriolar constrictor in kidneys from sham rats in the present study, during perfusion in vitro at a constant pressure, provides further indirect evidence that the open probability of KATP channels in afferent arteriolar smooth muscle is low under normal physiologic conditions. Less than 2% of the KATP channels in rabbit mesenteric arterial smooth muscle cells are estimated to be open in the absence of activators and in the presence of physiologic intracellular ATP (9), a situation that may also transpire in the renal afferent arteriole. However, our observation of afferent arteriolar dilation in response to structurally distinct KATP agonists such as pinacidil and PCO-400 in kidneys from sham rats confirms previous reports that this vascular segment is responsive to potassium channel openers (14, 15), and thus endowed with a recruitable pool of KATP channels.
In contrast to afferent arterioles from sham rats, vessels from STZ-treated rats exhibited significant constriction in response to glibenclamide. This response could not be attributed to an acute degenerative change of diabetic vessel function, because arteriolar diameter was stable in time-control experiments. It also seemed possible that the larger basal afferent arteriolar diameter in juxtamedullary nephrons of STZ-treated rats, characteristic of the hyperfiltration stage of IDDM (30), could have enhanced responsiveness to glibenclamide indirectly through stretch-induced changes in membrane potential and/or tangential wall stress. Because neither SNP nor nitric oxide has been reported to interfere with the glibenclamide binding to (or effects on) the KATP channel, the failure of glibenclamide to elicit constriction of SNP-dilated arterioles from sham rats makes it unlikely that the constrictor response to this agent observed in STZ rats is nonspecifically related to reduced arteriolar tone. The inability of glibenclamide to constrict SNP-dilated arterioles from sham rats is also consistent with the contention that the renal vasodilator response to nitric oxide does not involve opening of KATP channels (45). Overall, the responses to glibenclamide observed in the present study suggest that KATP channels are quiescent in normal rats but exert a vasodilator influence on afferent arteriolar tone during the hyperfiltration stage of diabetes.
We reasoned that a diabetes-induced opening of a significant component of the normal pool of afferent arteriolar KATP channels might fully explain the response to glibenclamide in STZ rats, thus reducing the population of closed KATP channels capable of responding to potassium channel openers. Deducing that this phenomenon should be indicated by a diminished vasodilator response to potassium channel openers, we were surprised to find that afferent arterioles from STZ rats exhibited exaggerated responses to these compounds. In spite of the larger baseline diameter in afferent arterioles from STZ rats, the dilator responses to both pinacidil and PCO-400 exceeded those of sham rats in terms of both percent change and absolute change in diameter. These observations suggest that the functional availability (expression or density) of afferent arteriolar KATP channels may be increased during the early stage of IDDM, or that the impact of these channels on membrane potential is exaggerated. The validity of these intriguing scenarios cannot be discerned from the results of the present study, requiring exploration at the electrophysiologic and molecular levels.
In addition to reversing renal vascular responses to potassium channel openers (15,39,46,47,48), glibenclamide inhibits afferent arteriolar dilator responses to glycolytic inhibition (15) and hypoxia (12). These observations indicate the potential for coupling afferent arteriolar smooth muscle metabolism to microvascular function via changes in KATP channel activity, as has been suggested to occur in a variety of vascular beds. The possible relevance of this putative metabolic coupling to regulation of afferent arteriolar tone in IDDM was first suggested by Marcus and coworkers (16), who documented diminished glucose uptake (associated with decreased expression of insulin-responsive GLUT4 transporter mRNA and polypeptide) in afferent arterioles from diabetic rats. Glucose transport is rate-limiting for glycolysis in vascular smooth muscle (49), and glycolytic inhibition evokes afferent arteriolar dilation through a glibenclamide-sensitive process (15). Accordingly, Marcus and coworkers (16) postulated that the effect of IDDM to decrease glucose uptake into afferent arteriolar smooth muscle might decrease cytosolic ATP levels, allowing opening of resident KATP channels, membrane hyperpolarization and reduced membrane excitability, closing of voltage-gated calcium channels, and vasodilation. It is also possible that KATP channel expression is promoted by the state of oxidative stress that is evident in the renal cortex even at this early stage of IDDM (50). Although the precise underlying mechanism remains to be established, our observation that glibenclamide restored afferent arteriolar diameter in STZ rats to a value not different from that observed in sham rats supports the contention that opening of KATP channels may be a primary factor contributing to the afferent arteriolar dilation that underlies diabetic hyperfiltration. However, we note that a recent study failed to discern any influence of the putative KATP blocker U37883A on renal hemodynamics and GFR in anesthetized rats with STZ-induced IDDM (41). Because of the potential indirect (systemic) influences on renal function in clearance studies performed in anesthetized rats, the problems inherent with in vitro studies, of arteriolar function, and uncertainities about the efficacy and specificity of the pharmacologic agents used in these studies, further investigation is required to more fully evaluate the role of afferent arteriolar KATP channels in engendering diabetic hyperfiltration.
One final observation deserves comment, although it is somewhat tangential to the original aims of the study. The enormous dilation evoked by pinacidil in afferent arterioles from STZ rats refutes our previous suggestion (offered on the basis of diminished responses to supramaximal concentrations of SNP) that afferent arterioles are nearly maximally dilated during diabetic hyperfiltration (30). The magnitude of the dilator response to pinacidil observed in STZ kidneys is substantially greater than afferent arteriolar responses to any dilator stimulus that we have imposed on in vitro blood-perfused juxtamedullary nephrons. We cannot yet distinguish between the possible involvement of specific pathophysiologic alterations in electromechanical coupling events or the role of arteriolar hypertrophy in this phenomenon, although we note that renal hypertrophy is already evident at this stage in our IDDM model.
In summary, experiments were performed to test the postulate that the impact of KATP channels on renal afferent arteriolar tone is exaggerated during the hyperfiltration stage of IDDM. Glibenclamide did not alter afferent arteriolar diameter in normal (sham) rats, although potassium channel openers (pinacidil and PCO-400) evoked afferent arteriolar dilation, supporting the contention that these vessels are endowed with KATP channels that are normally quiescent. In contrast, 2 wk after induction of IDDM, glibenclamide reduced afferent arteriolar diameter (to values similar to those observed in normal rats), suggesting that open KATP channels exert an arteriolar dilator influence under these conditions. Moreover, the dilator responses to pinacidil and PCO-400 were exaggerated in kidneys from diabetic rats, consistent with an increased functional expression of KATP channels and/or an enhanced impact of these channels on membrane potential and, hence, arteriolar tone. We conclude that the early stage of IDDM in the rat is associated with the apparent emergence of a KAPT-dependent vasodilator influence on renal afferent arteriolar tone, the consequences of which can be expected to contribute to diabetic hyperfiltration.
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