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1 Departments of Medicine and Physiology and Biophysics, Division of Nephrology, Mayo Clinic and Foundation, Rochester, Minnesota 55905, and 2 Department of Cardiac Surgery, University of Heidelberg, IM Neuenheimer Feld 110 Heidelberg 69115, Germany
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The role of nitric oxide (NO) in the
regulation of the intrarenal microcirculation in streptozotocin
(STZ)-induced diabetes mellitus in rats is not clear. We examined renal
cortical and papillary hemodynamics in STZ rats and determined the
effects of systemic inhibition and stimulation of NO synthesis. Renal blood flow in cortical (QCC),
and inner medullary ascending
(QAV) and descending
(QDV) vasa recta capillaries was
measured by fluorescence videomicroscopy in STZ Munich-Wistar rats and
nondiabetic control rats. Ten days after STZ injection (80 mg/kg ip),
basal QCC and QDV were significantly greater in
STZ rats (n = 16) compared with control rats (n = 15). Infusion of
NG-monomethyl-L-arginine
(L-NMMA, 15 mg/kg bolus, 500 µg · min
1 · kg1
iv) decreased QCC (
41%),
QAV (38%), and
QDV (37%) in control rats
(n = 6) and to a significantly greater
magnitude than in STZ rats (n = 7),
QCC (14%),
QAV (20%), and
QDV (25%). Coinfusion of
L-arginine
(L-Arg, 1 mg · kg1 · min1
iv) with L-NMMA increased
QCC to a significantly greater
extent (P < 0.01) in control rats
compared with STZ rats. In subsequent studies, infusion of
L-Arg alone increased
QCC (+50%),
QAV (+16%), and
QDV (+11%) in control rats
(n = 5) but had no effect in STZ rats
(n = 5). These results show that the
response of renal cortical and papillary capillary blood flow to both
inhibition and stimulation of NO synthesis is attenuated in the early
onset of STZ-diabetes mellitus rats compared with control rats.
experimental insulin-dependent diabetes mellitus; nitric oxide-dependent renal vasodilation; diabetic endothelial dysfunction; NG-monomethyl-L-arginine; videomicroscopy
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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NITRIC OXIDE (NO)-dependent vasodilation has been shown to be an important factor in the maintenance and regulation of vascular tone in the renal microcirculation (6, 20, 21, 31, 34). Evidence that glomerular arteriolar resistances are regulated by basal NO levels is supported by observations of vasoconstriction in afferent and efferent arterioles of both superficial cortical (20, 21) and juxtamedullary nephrons (34) following NO synthesis inhibition. Both cortical and medullary renal blood flows have been shown to decrease with systemic inhibition of NO in nondiabetic rats (20, 21, 31, 34).
The renal microcirculation after the onset of insulin-dependent type 1 diabetes mellitus is characterized by glomerular hyperfiltration and vascular dysregulation (7, 11, 16). Renal hyperperfusion occurs after the early onset of diabetes mellitus and depends on the extent of hyperglycemia and insulin treatment (16). Dysfunction of the diabetic renal vasculature is apparent in an increased sensitivity to renal vasoconstrictors, such as adenosine, radiocontrast-media agents, and vasoconstrictor release during cardiac surgery (1, 25-27). This vascular dysregulation in diabetes mellitus has been attributed to numerous factors, including changes in vasoactive mediator release and a diminished NO-dependent vasodilation (11, 25, 26). There is considerable evidence suggesting that NO-dependent renal vasodilation is impaired in chronic states of diabetes mellitus, and it has been proposed that this may account for the higher susceptibility of the diabetic kidney to vasoconstrictor stimuli and the development of diabetic nephropathy (7, 9, 11, 25, 32).
Hence it has been hypothesized that renal NO-dependent vasodilation may be defective in diabetes despite increased NO generation. However, no in vivo study has been performed to determine the role of NO-dependent renal vasodilation in early onset of experimental diabetes mellitus by direct measurements of renal cortical and medullary papillary microcirculations. Therefore the present study was performed to determine 1) whether basal renal cortical and medullary papillary blood flow hemodynamics differ in the early onset of streptozotocin (STZ)-induced diabetes mellitus compared with nondiabetic control rats and 2) the role of NO-dependent vasodilation of the cortex and papilla in the renal microcirculation in the early onset of STZ-diabetes mellitus.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animal Preparation
All experiments in the present study were conducted on young (5-6 wk, 100-135 g body wt) Munich-Wistar rats purchased from Harlan Sprague Dawley, Indianapolis, IN, and fed a normal Purina rat chow containing 0.1 mM sodium/g. The animals were allowed free access to water but were deprived of food overnight immediately before the experiment. The animals were prepared for videomicroscopy as follows. They were anesthetized with an intraperitoneal injection of 100 mg/kg body wt Inactin (thiobutabarbital, Byk Gulden) and placed on a heated table to maintain rectal temperature at 36-38°C. After tracheal cannulation with polyethylene (PE)-200 tubing, two PE-50 catheters were inserted for infusions, one into the left jugular vein and the other into the right jugular vein. Arterial blood pressure was continuously monitored via a left carotid artery catheter connected to a Statham pressure transducer. The arterial catheter was filled with normal isotonic saline, containing 30 IU/ml heparin to prevent clotting at the tip of the catheters. The heart rate was derived from the pressure tracing connected to a thermoprinter (WK-280 R; Foehr Medical Instruments). Two intravenous infusions were started: 1) 2.5% inulin in 0.9% isotonic saline at 20 µl · min1 · 100 g body wt1 and
2) 5% bovine serum albumin (Sigma
Chemical) in saline at 20 µl · min1 · 100 g body wt1. Although the
first infusion was given for the duration of the experiments, the
albumin infusion was given only for 1 h during surgery to replace
surgical fluid losses. The left kidney was exposed by an abdominal
midline incision. Urine samples for clearances were obtained by
cannulating the right ureter. The left kidney was carefully freed from
perirenal tissue and placed on a mineral oil-soaked cotton pillow in a
Lucite holder to minimize motion artifact. Care was taken not to
stretch the renal artery or vein. Young Munich-Wistar rats are commonly
used in investigations of medullary hemodynamics because of their
relatively long papillae, which extrude out of the renal pelvis into
the ureter (20). This papillary tip was exposed for the vasa recta
studies by excising the ureter from the kidney. Recordings were made
from two areas near the base of the exposed papilla. Except during vasa
recta recordings, the papilla was covered with plastic cling wrap to prevent drying. In the cortical hemodynamic studies, capillaries were
visualized from the surface of the kidney. Between each recording period the whole kidney was covered with plastic cling wrap to prevent
evaporation. On completion of the experiment, all animals were killed
by an overdose of pentobarbital.
Experimental Insulin-Dependent Diabetes Mellitus
The animal model of insulin-dependent diabetes mellitus (IDDM) type 1 was achieved by an intraperitoneal injection of 80 mg/kg STZ (Sigma) dissolved in sodium citrate buffer (pH 4.2) in young Munich-Wistar rats (4-5 wk, 80-90 g body wt). Three days after STZ injection and on the day of the experiment, blood glucose levels were measured from tail blood samples. Animals with a blood glucose below 260 mg/dl were not included in the experimental series. The experiments started 8-12 days after STZ administration without insulin treatment, and nondiabetic age-matched rats served as controls. The untreated 80 mg/kg STZ model of IDDM was chosen to reduce malnourishment, catabolic state, weight loss, and hyperphagia, since the administered dose of STZ does not completely destroy all beta islet cells of the rat pancreas (28), and a remaining low basal level of insulin production is achieved with this IDDM model. Furthermore, moderate hyperglycemia was stable until the day of the experiment (blood glucose levels 3 days after STZ, 296 ± 20 mg/dl; on the day of the experiment, 328 ± 35 mg/dl).Fluorescence Videomicroscopy of Superficial Cortical and Vasa Recta Capillaries
The direct comparison of blood flow in the microcirculations of cortex and medulla by visualization of fluorescent-labeled red blood cells was made possible by applying the videomicroscopy techniques previously used for measurement of blood flow in the superficial capillaries of the cortex and the papillary vasa recta (20, 34).Capillary blood flow was determined by measuring red blood cell velocity in superficial cortical and vasa recta capillaries visualized at a ×1,000 magnification by means of fluorescence videomicroscopy. This method is based on the dual-slit tracking technique as modified by Gussis et al. (14) for measurement of red blood cell velocity in the mammalian inner medulla.
The methodology followed in these studies, including the fluorescein
isothiocyanate (FITC) labeling of red blood cells (35) and
-globulin, is fully described elsewhere (15). In brief, 0.2 ml of
the FITC-bound -globulin and 0.05 ml of the labeled red blood cells
were injected intravenously.
The papilla or cortex was illuminated with fluorescent light under the objective (magnification, ×25; numerical aperture, 0.35) of an epifluorescence microscope (Leitz, Wetzlar), and the image was transferred by a silicone-intensified television camera (C2400 Hamamatsu, Photonic Microscopy) to a high-resolution monitor. The televised images of two areas of the microcirculation were each recorded for 2-4 min on videotape; the recordings were made during the first 15 min of the three experimental periods and analyzed later as follows. Two photometric windows were positioned 7-26 µm apart over a single vessel. Only flowing capillaries were measured, nontortuous vessels being selected in preference. Of these, only relatively straight regions of the vessels were studied. If a capillary was seen to have more than one branch, only the main branch or one of its subsidiaries was measured. At the base of the papilla, the vasa recta are generally straight and parallel, aiding vessel selection. The superficial cortical capillaries, on the other hand, form a more tortuous network. Care was taken that measurements were not made from different regions of the same capillary. Camera rotation enabled the monitor picture to be orientated such that the capillary region of immediate interest was situated vertically to enable measurement.
Red blood cell velocity was calculated by means of a cross-correlation software package (model 204C, Instruments for Physiology and Medicine) from the time delay between fluctuations in light intensity detected by the windows. The value typically represented an average of 200 or more velocity measurements per 2-min period. Vessel diameter was also measured by means of a caliper (smallest division 0.1 mm) from stopped-frame television images and corrected for the final magnification of the monitor image (×1,000). Mean vessel blood flow (Vblood) was calculated by using the equation derived empirically by Zimmerhackl et al. (35) from studies in which blood of different hematocrits was perfused through quartz capillary tubes
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Single capillary blood flow (QC) of both superficial cortical (QCC) and vasa recta [ascending vasa recta flow (QAV) and descending vasa recta flow (QDV)] capillaries was then computed according to the equation
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Experimental Protocol
The experiment was started after a 60-min recovery period following surgery. Subsequently three experimental periods, each lasting 30 min, were carried out. In each experimental period, renal clearance studies were performed and renal cortical and papillary blood flows were determined by recording 2-4 min each. A midpoint blood sample was withdrawn via the carotid artery cannula for inulin and electrolyte measurements in each clearance period.Experimental Groups
Group 1: Effect of systemic infusion of
L-NMMA and coinfusion of
L-NMMA and
L-arginine on renal
QCC and papillary
QAV and QDV in
nondiabetic control rats (n = 6).
In the first clearance period, the effects of an isotonic normal saline
infusion (13 µl/min) on renal cortical and papillary blood flow were
determined. On completion of the first clearance, NG-monomethyl-L-arginine
(L-NMMA) was administered
intravenously as a bolus injection of 15 mg/kg body wt followed by an
infusion of 500 µg · min1 · kg
body wt1 in
groups 1 and
2. Ten minutes after starting the
L-NMMA infusion, a second
clearance period was started, during which cortical capillaries and
vasa recta recordings were made and a blood sample was collected. Thereafter, in the third clearance, effects of coadministration of
L-NMMA (500 µg · min1 · kg
body wt1 iv) and
L-arginine
(L-Arg, 1 mg · min1 · kg
body wt1 iv) on renal
cortical and papillary blood flow were determined correspondingly.
Group 2: Effect of systemic infusion of L-NMMA and coinfusion of L-NMMA and L-Arg on renal QCC and papillary QAV and QDV in STZ-diabetic rats (n = 7). The protocol of this group was identical to group 1 but was performed in STZ rats.
Group 3: Effect of systemic infusion of
L-Arg on renal
QCC and papillary
QAV and QDV in
nondiabetic control rats (n = 5).
In the first and second clearance periods, the effects of an isotonic
saline infusion (13 µl/min) on renal clearance and cortical and
papillary blood flow were determined. Thereafter, in the third clearance, L-Arg was
coadministered intravenously by 1 mg · min1 · kg
body wt1. Ten minutes after
starting L-Arg infusion the
third clearance period was started and renal clearance and cortical and
papillary blood flow were determined correspondingly.
Group 4: Effect of systemic infusion L-Arg on renal QCC and papillary QAV and QDV in STZ-diabetic rats (n = 5). The protocol of this group was identical to group 3 but was performed in STZ rats.
Group 5: Effect of time and systemic infusion of normal saline vehicle (Veh) on renal QCC and papillary QAV and QDV in nondiabetic control rats (n = 4). Groups 5 and 6 served as vehicle-time controls with a continuous infusion of the isotonic saline vehicle infusion (13 µl/min) throughout all three clearance periods. In all three clearance periods only the effects of the isotonic saline infusion vehicle on renal cortical and papillary blood flow were determined.
Group 6: Effect of time and systemic infusion of Veh on renal QCC and papillary QAV and QDV in STZ-diabetic rats (n = 4). The protocol of this group was identical to group 5 but was performed in STZ rats.
Analytic Methods
Blood glucose levels were measured with a blood glucose meter (One Touch, Lifescan) 3 days after STZ injection and on the day of the experiment after the equilibration period. Glomerular filtration rate (GFR) was calculated based on the clearance of inulin. Inulin in plasma and urine was measured by the Anthrone method (12). Sodium concentrations in plasma and urine were measured by using a flame photometer (IL943 Flame Photometer, Instrumentation Laboratory). Urinary and plasma total NO (NO2/NO3) were measured by a colorimetric nonenzymatic NO assay kit (Oxford Biomedical Research). A thermoprinter (General Scanning) was used for
continuous blood pressure recording via a Statham transducer.
Statistical Analysis
All values were expressed as means ± SE. For the estimation of capillary blood flow, 11-16 ascending vasa recta, 10-16 descending vasa recta, and 14-26 cortical capillaries were measured per animal, expressed as N for measurements per capillary, with each vessel serving as its own control. The data were then averaged for each period, and from these averages the means ± SE was calculated for each group. Two-factor analysis of variance (ANOVA) with repeated measures on one and unpaired Student's t-test were performed as appropriate. Significance was considered with P < 0.05.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Basal Renal Hemodynamics of Cortical and Papillary Renal Blood Flow in Early Onset of STZ-Induced Diabetes Mellitus in Rats
To determine basal renal hemodynamics, we pooled the data of the vehicle period (Veh) in control rats (groups 1, 3, and 5) and STZ rats (groups 2, 4, and 6); the pooled data are presented in Table 1. In brief, baseline cortical capillary blood flow was significantly greater (P < 0.01) in STZ rats (3.1 ± 0.1 nl/min; n = 16, N = 64) compared with control rats (2.6 ± 0.1 nl/min; n = 15, N = 56). Medullary capillary blood flow of descending vasa recta was significantly greater (P < 0.05) in STZ rats (12.4 ± 1.0 nl/min; n = 16, N = 37) compared with control rats (9.6 ± 0.7 nl/min; n = 15, N = 37). Medullary capillary blood flow of ascending vasa recta tended to be higher in STZ rats (9.4 ± 0.5 nl/min; n = 16, N = 39) compared with control rats (8.6 ± 0.4 nl/min; n = 15, N = 44) but was not significantly different (P = 0.07). Basal GFRs of the right kidney were significantly greater (P < 0.05) in STZ rats (0.9 ± 0.09 ml/min; n = 16) compared with control rats (0.7 ± 0.06 ml/min; n = 15). Basal total urinary NO and NO excretion tended to be higher in STZ rats (urinary NO2/NO3 excretion, 125.0 ± 38.4 pmol/min) compared with control rats
(urinary NO2/NO3
excretion, 74.6 ± 18.9 pmol/min; P = 0.06). Total plasma NO was significantly higher in STZ rats (12.0 ± 1.4 µM) compared with control rats (7.2 ± 0.4 µM,
P < 0.05).
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Effect of Systemic Infusion of L-NMMA on Renal Cortical and Papillary Capillary Blood Flow
The renal hemodynamic responses to systemic infusion of L-NMMA are summarized in Tables 2 and 3. Systemic infusion of L-NMMA increased mean arterial pressure in control and STZ rats. L-NMMA decreased diameter, red blood cell velocity, and blood flow of cortical, ascending, and descending vasa recta capillaries in control rats. In STZ rats, however, L-NMMA did not change the diameter, red blood cell velocity, and blood flow of cortical capillaries. In STZ rats, L-NMMA only decreased the diameter of ascending from 22 ± 0.6 to 20.5 ± 0.6 µm and decreased blood flow only in descending vasa recta.
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In Fig. 1, the
L-NMMA-induced reductions of
renal blood flow are presented as percent renal blood flow reduction
(
Q%) because of different baseline values between the different
capillaries (cortical, ascending, and descending vasa recta
capillaries) and between the different groups (control and STZ, Fig.
1). The decrease in renal blood flow in cortical, ascending, and
descending vasa recta capillaries was significantly greater in control
rats compared with STZ rats (P < 0.05, P < 0.01, P < 0.0001, respectively, see Fig.
1).
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The administration of L-Arg reversed the L-NMMA-induced decreases in vessel diameter, red blood cell velocity, and renal blood flow of cortical and ascending vasa recta capillaries of control rats. The percent increase of renal cortical capillary and descending vasa recta capillary blood flow by L-Arg was significantly greater in control rats compared with STZ rats (P < 0.05, P < 0.05, respectively; see Fig. 1).
Effect of L-Arg on Renal Cortical and Papillary Capillary Blood Flow
The renal hemodynamic data of systemic infusion of L-Arg alone are summarized in Tables 4 and 5. Systemic infusion of L-Arg increased the diameter of cortical and ascending vasa recta capillaries and increased renal blood flow in cortical, ascending, and descending vasa recta capillaries of control rats.
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In contrast, in STZ rats, systemic infusion of
L-Arg had no effect on vessel
diameter or renal blood flow of cortical, ascending, and descending
vasa recta capillaries. The percent increase of renal blood flow
(Q%) by systemic infusion of
L-Arg was significantly greater
in cortical and descending vasa recta capillaries of control rats
compared with STZ rats (P < 0.01, P < 0.01, respectively) as
demonstrated in Fig. 2.
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Time and Vehicle Controls
The renal hemodynamic data of systemic infusion of Veh are summarized in Tables 6 and 7. There were no time-dependent effects on renal cortical and papillary hemodynamics in control and STZ rats with systemic infusion of Veh with respect to vessel diameter, red blood cell velocity, capillary blood flow, GFR, or fractional sodium excretion.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Renal Cortical and Papillary Capillary Blood Flow in Early Onset of STZ-Diabetes Mellitus in Rats
In the current studies, basal cortical capillary and papillary renal blood flows were higher in the early onset of STZ-diabetic rats compared with control rats. These findings are consistent with previous studies that have demonstrated increased renal blood flow in STZ rats compared with control rats (3, 16, 30). The GFRs of contralateral kidneys of STZ rats were increased (group 2) or tended to increase (groups 4 and 6) as has been reported previously (3, 16, 30). In experiments with a similar protocol, clearances obtained from the contralateral kidney are similar to those from the kidney in which renal blood flow is studied (20, 24).Inasmuch as urinary and plasma
NO2/NO3
tended to be elevated in STZ rats compared with control rats, increased
renal NO generation may be in part responsible for increased vasa recta
and cortical capillary blood flow in STZ rats. However, the diabetic
kidney may not be the only source for the increased urinary
NO2/NO3 since plasma
NO2/NO3
was increased in STZ rats. Hyperphagia in STZ rats may contribute to an
increased plasma and urinary
NO2/NO3 concentration; this, however, seems to be a minor factor because rats
were fasted overnight. In agreement with our findings, several studies
have found an increased generation of renal NO in experimental diabetes
independent of dietary intake (3, 30).
The renal hyperperfusion, which occurs in the early onset of diabetes, is thought to be caused by an increased renal vasodilation in diabetes due to increased generation of renal vasodilators such as NO (3, 30). This hyperperfusion is thought to be one factor leading to the diabetic hyperfiltering kidney and the long-term vascular and interstitial tissue injury manifest in diabetic nephropathy (19). However, the concept that the diabetic kidney generates more NO leading to increased renal vasodilation, renal hyperperfusion, perfusion injury, and consequent diabetic nephropathy remains controversial.
Effects of Inhibition and Stimulation of NO Synthesis on Renal Cortical and Papillary Hemodynamics in Control and STZ-Diabetic Rats
In control rats, L-NMMA decreased and L-Arg increased both cortical and papillary capillary blood flows. Numerous studies have shown that inhibition of NO synthesis decreases renal blood flow in control rats due to vasoconstriction in afferent and efferent arterioles, and in medullary capillaries (6, 20, 34). In agreement with these studies, L-NMMA reduced the vessel diameter, red blood cell velocity, and blood flow in cortical and papillary capillaries in control rats. Stimulation of NO generation with L-Arg reversed the effects of L-NMMA-induced vasoconstriction on capillary diameter, red blood cell velocity, and blood flow of control rats. A similar response was reported in isolated descending vasa recta of the outer medulla, in which inhibition of NO enhanced norepinephrine-induced vasoconstriction and L-Arg reversed this effect (33).Like afferent arterioles, efferent arterioles possess smooth muscle
cells (2). In descending vasa recta, the smooth muscle cells are
gradually replaced by pericytes forming an incomplete layer of
contractile cells. It was shown that the pericytes surrounding the
descending vasa recta within the outer medulla contain smooth muscle
-actin mRNA and protein and are therefore the site of contractile
elements (23). However, descending vasa recta of the inner medulla do
not contain contractile pericytes (23). Because the descending vasa
recta segments studied in the current study represent the inner
medulla, and therefore do not contain contractile elements, the
decreases in diameter of these capillary segments reflect upstream
vascular responses. In conclusion, cortical and papillary capillaries
respond similarly to inhibition and stimulation of NO synthesis in
control rats.
Despite increased basal renal blood flow, the present findings show an attenuated response of the diabetic renal cortical and papillary microcirculation to both NO synthesis inhibition and stimulation of NO generation in STZ rats compared with control rats. The magnitude by which L-NMMA decreases both cortical and papillary capillary blood flows was significantly greater in control rats than in STZ rats. In STZ rats, L-NMMA did not change vessel diameter of cortical and descending vasa recta capillaries. Likewise, the magnitude by which L-Arg increased renal papillary and cortical capillary blood flows was markedly attenuated in STZ rats compared with control rats. Furthermore, in contrast to control rats, cortical capillaries and vasa recta of STZ rats did not vasodilate with systemic infusion of L-Arg.
Analysis of the capillary blood flow in STZ rats suggests that there is
a regional difference in the degree of the
L-NMMA response, being greatest
in descending capillaries (25% renal blood flow reduction) more than
ascending capillaries (20% renal blood flow reduction) more than
cortical capillaries (14% renal blood flow reduction). In
STZ rats, the L-NMMA-induced
reduction of capillary blood flow was significant only in descending
capillaries, and hence the unresponsiveness of NO synthesis inhibition
was greatest in cortical capillaries. In control rats, medullary NO production was shown to be higher compared with NO production of the
cortex (36). Our current observations indicate that NO generation is
normal or somewhat increased in STZ compared with control rats based on
plasma and urinary
NO2/NO3 concentrations, consistent with previous studies (3, 30). Hyperglycemia
may be a contributing factor for this observation because high glucose
was shown to increase NO generation via stimulation of endothelial
constitutive NO synthase (8). Therefore, in STZ rats higher NO
production in papillary capillaries may account for the somewhat
maintained response to L-NMMA in
the papillary microcirculation compared with the cortical microcirculation.
It could be hypothesized that increased basal NO generation and NO-dependent renal vasodilation in the diabetic renal vasculature may require higher doses of NO inhibition and stimulation to have the same effect on cortical and vasa recta capillary blood flow as in control rats. However, the following observations are not in support of this hypothesis. The systemic doses of the NO synthesis inhibitor L-NMMA and stimulator of NO synthesis L-Arg were high (20) and were effective in the extrarenal systemic diabetic vasculature because mean arterial pressure increased with L-NMMA and decreased with L-Arg in both STZ and control rats. The changes in cortical capillary diameter with inhibition and stimulation of NO synthesis occurred in control but not STZ rats. Also, Ohishi and Carmines (22) have found that the constriction of afferent arterioles in response to L-NMMA was blunted in diabetic rats. This suggests that the diabetic cortical microvasculature is insensitive to NO-dependent vasodilation.
Several mechanisms could account for a reduced responsiveness of the diabetic renal vasculature to NO-dependent vasodilatation: 1) inactivation of NO and/or 2) a reduced sensitivity of the smooth muscle cells to NO despite normal or increased generation of NO, 3) diminished autoregulatory adjustments in renal vascular resistance, 4) baroreflex-mediated alterations in renal sympathetic nerve activity, and 5) increased renal vasodilation by NO-independent factors (prostaglandin), hence offsetting the effects of NO synthesis inhibition or stimulation.
Despite increased NO synthesis in diabetes mellitus, numerous studies have found an impairment of NO-dependent vasodilation in diabetes mellitus (5, 7, 9, 11, 21, 22, 25, 32), and glucose was shown to impair endothelium-dependent relaxation (29). These studies suggest that several factors, including oxygen-derived free radicals, increased production of NO antagonists such as endothelin 1, and quenching of NO by advanced glycosylation end-products, inactivate NO and therefore are responsible for a diminished NO-dependent vasodilation in diabetes (4, 11, 18, 22).
Inactivation of NO during upregulated NO synthesis, e.g., increased destruction of NO (11), could well explain a diminished guanosine 3', 5'-cyclic monophosphate (cGMP)-coupled vasodilation in diabetes, since a reduced capacity to generate NO-dependent cGMP has been reported in glomeruli of STZ rats (9, 32). Hence increased NO synthesis may not result in an increased cGMP-coupled smooth muscle relaxation.
Another mechanism of a dysfunctional NO-dependent renal vasodilation in
diabetes independent of NO generation or inactivation could be a
defective cGMP-coupled smooth muscle cell relaxation. Numerous factors
may be responsible for this mechanism, including decreased
transport/delivery of NO and decreased responsiveness of the smooth
muscle cell to NO (11). Dai et al. (10) found impaired vasodilation in
response to acetylcholine in renal arteries of STZ-diabetic rats, which
increased with the duration of the diabetes. In another study (19), the
administration of the NO-donor glyceryl trinitrate did not increase
renal plasma flow in STZ rats in contrast to control rats. In this
study, STZ rats had a markedly increased urinary
NO2/NO3 excretion compared with control rats despite the reduced responsiveness to the NO donor.
On the other hand, NO-independent factors could account for the present findings, such as an impaired renal vascular autoregulation in STZ rats. If autoregulation is impaired in STZ rats (even if the renal NO system is normal), the increase in arterial pressure caused by L-NMMA would promote an increase in renal blood flow and GFR, thus tending to offset any renal vasoconstriction that might result from inhibition of the direct NO-dependent influence on the renal vasculature. The result would be a smaller L-NMMA-induced decrease in renal blood flow in STZ rats as seen in the current observations.
Taken together, the present findings show an increased basal blood flow in renal cortical and descending vasa recta capillaries in the early onset of STZ-induced diabetes mellitus in rats. STZ rats had an attenuated responsiveness to inhibition and stimulation of NO synthesis in both the cortical and papillary renal microcirculations. We conclude that NO-dependent renal vasodilation is unresponsive in both renal cortical and to a lesser degree in papillary microcirculations in the early onset of STZ-diabetes due to a defective NO-dependent vasodilation or a diminished renal vascular autoregulation.
This vascular dysfunction of the renal NO-dependent vasodilator capacity in diabetes may contribute to the higher susceptibility of diabetic kidney to vasoconstrictor stimuli (25, 26). Accordingly, in clinical situations, the release of renal vasoconstrictors following radiocontrast media (27) and during extracorporeal circulation in open-heart surgery (1) may increase the risk of acute renal failure (27) and papillary necrosis (13) in diabetic patients.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the support of John Haas, Jennifer M. Gross, and Dr. Carla Ramsey, and the secretarial assistance of Joanne Zimmerman.
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FOOTNOTES |
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The study was supported by the Department of Cardiac Surgery, University of Heidelberg, by the National Heart, Lung, and Blood Institute Grant HL-55594, and by the Mayo Foundation.
Part of this study was presented at the annual meeting of the American Society of Nephrology, San Antonio, TX, 1997, and Philadelphia, PA, 1998.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: F. G. Knox, Depts. of Medicine, and Physiology and Biophysics, Guggenheim 9, Mayo Clinic, Rochester, MN 55905 (E-mail: Pflueger.Axel{at}mayo.edu).
Received 29 January 1999; accepted in final form 6 May 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Ashraf, S.,
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