Afferent Diameter Research Articles

Abstract P056: MHC CLass II-associated Invariant Peptide (CLIP) Antagonism During Chronic Lipopolysaccharide Treatment Preserves Afferent Arteriolar Autoregulatory Behavior

Lipopolysaccharide (LPS) is a cell wall component of gram-negative bacteria that can activate toll-like receptor 4, which in turn, activates the innate immune system. Chronic immune system activation is linked to blunted afferent arteriolar autoregulatory behavior and kidney injury. MHC Class II-associated invariant peptide (CLIP) provides a critical step in antigen processing, presentation, and adaptive immune system activation. Accordingly, we postulated that treatment with a competitive CLIP antagonist (CAP) during chronic low-dose LPS exposure would preserve afferent arteriolar autoregulatory behavior. Rats were implanted with osmotic minipumps (day 0) for infusion of LPS (0.01mg/kg/day) or saline (0.9% NaCl; 0.5μl/hr) for 8 days and then the kidneys were harvested for juxtamedullary nephron studies. Four groups (n=6/group) were studied: Control, LPS, LPS + CAP and Sham + CAP. Both LPS + CAP and Sham + CAP groups were treated with CAP (3mg/kg/day; i.p.) on days 1-7. Autoregulatory behavior was assessed in these groups by increasing perfusion pressure in 15 mmHg increments from 65 to 170 mmHg. Starting baseline diameters were similar across the control (15.2 ± 1.2 μm), LPS (14.1 ± 1.0 μm), LPS + CAP (15.1 ± 1.0), and Sham + CAP (15.7 ± 0.6) groups. When perfusion pressure was increased from 65 to 170 mmHg, control and sham + CAP afferent arteriolar diameter decreased significantly by 26 ± 4% and 25 ± 2% (P<0.05), respectively. In contrast, afferent diameters from LPS treated kidneys decreased by just 5 ± 2% over the same pressure range indicating impaired afferent arteriolar autoregulatory behavior. In LPS + CAP treated kidneys, afferent arteriolar diameter decreased by 17 ± 2% (P<0.05) over the same pressure range signifying preservation of autoregulatory behavior. These data support the hypothesis that CLIP antagonism during chronic low dose LPS treatment preserves afferent arteriolar autoregulatory behavior. Inhibiting CLIP may open novel therapeutic targets for inflammatory kidney disease.

Abstract MP15: Afferent Arteriolar Endothelial-dependent Dilation is Impaired Prior to the Development of Radiation-induced Nephropathy and Hypertension

Chronic kidney disease (CKD) occurs in 15% of hematopoietic stem cell transplant (HSCT) patients, and has been clearly linked to irradiation at the time of the HSCT. CKD due to radiation also occurs after radionuclide therapy for cancer. Radiation nephropathy is expressed in rats and in humans as proteinuria, azotemia, and hypertension. There is a latent period of 6-8 weeks after irradiation to the development of proteinuria, azotemia, and hypertension in rats. This study tested the hypothesis that afferent arteriolar responses to the endothelial-dependent dilator acetylcholine are impaired prior to the development of proteinuria, azotemia, and hypertension in rats exposed to total body irradiation (TBI). Male WAG/RijCmcr rats were subjected to TBI (11 Gy) and afferent arteriolar responses to acetylcholine using the juxtamedullary nephron technique were determined at one, three, and six weeks. Systolic blood pressure (117 ± 6 vs. 119 ± 4 mmHg) and BUN (15.6 ± 1.4 vs. 15.8 ± 0.8 mg/dl) were not different between control and TBI groups at 6 weeks. Afferent arteriolar diameters averaged 22.5 ± 0.8 μm (n=30) in controls and 21.7 ± 0.7 μm (n=27) in TBI rats and were not different between control and TBI groups at 1, 3, or 6 weeks. Acetylcholine dilator responses were progressively attenuated from one to six weeks in TBI compared to control rats. During exposure to acetylcholine (0.01, 0.1, 1, and 10 μmol/L), afferent diameter increased by 8 ± 2%, 18 ± 3%, 27 ± 2% and 31 ± 3% in control rats (n=6), and 3 ± 2%, 9 ± 3%, 12 ± 3%, and 16 ± 3% in kidneys of 3 week TBI rats (n=8, p<0.05). However, TBI did not change the norepinephrine-mediated vasoconstrictor responses or the nitroprusside vasodilator responses at 1, 3, or 6 weeks. Renal microvessels were isolated from control and TBI groups at 3 and 6 weeks for protein expression assessment of endothelial enzymatic pathways Cyp2C, COX, and NOS. We found that epoxygenase Cyp2C23 and Cyp2C11 protein expressions were significantly lower 3 and 6 weeks following TBI compared to control rats. Taken together, these results indicate that the impaired afferent arteriolar endothelial-dependent acetylcholine responses and decreased epoxygenase enzymes precede the development of proteinuria, azotemia, and hypertension in irradiated rats.

Role of connexin 45 in the autoregulatory response of the afferent arteriole

Connexins expressed in renal arterioles are believed to influence the regulation of arteriolar tonus and renal blood flow. Connexin 45 (Cx45), which is primarily expressed in vascular smooth muscle cells of the renal afferent and efferent arterioles, has been suggested to play a role in the regulation of renin secretion. Also, Cx45 may be involved in the transmission of the signal initiating the tubuloglomerular feedback (TGF) response.In mice lacking Cx45 expression in vascular smooth muscle cells we examined the renal autoregulatory response by measuring changes in afferent arteriolar diameter in response to step increases in perfusion pressure. We used the isolated blood‐perfused juxtamedullary nephron preparation to visualize afferent arterioles. Cx45 deficient mice did not seem to have a higher mean arterial blood pressure than age‐matched litter‐mate wild type controls. The reduction in afferent arteriole diameter in response to step increases in renal perfusion pressure was significantly reduced in Cx45 deficient mice compared to the wild‐type litter‐mates. Removal of the TGF response did not seem to affect the ability to regulate afferent diameter in Cx45 deficient animals compared to wild‐type litter mates. These results suggest that Cx45 plays a major role in the myogenic component of the autoregulatory response of the afferent arteriole.

Tempol Prevents Altered K + Channel Regulation of Afferent Arteriolar Tone in Diabetic Rat Kidney

Experiments were performed to test the hypothesis that oxidative stress underlies the enhanced tonic dilator impact of inward-rectifier K(+) channels on renal afferent arterioles of rats with streptozotocin-induced diabetes mellitus. Sham and diabetic rats were left untreated or provided Tempol in their drinking water for 26±1 days, after which afferent arteriolar lumen diameter and its responsiveness to K(+) channel blockade were measured using the in vitro blood-perfused juxtamedullary nephron technique. Afferent diameter averaged 19.4±0.8 μm in sham rats and 24.4±0.8 μm in diabetic rats (P<0.05). The decrease in diameter evoked by Ba(2+) (inward-rectifier K(+) channel blocker) was 3 times greater in diabetic rats than in sham rats. Glibenclamide (K(ATP) channel blocker) and tertiapin-Q (Kir1.1/Kir3.x channel blocker) decreased afferent diameter in diabetic rats but had no effect on arterioles from sham rats. Chronic Tempol treatment prevented diabetes mellitus-induced increases in both renal vascular dihydroethidium staining and baseline afferent arteriolar diameter. Moreover, Tempol prevented the exaggeration of afferent arteriolar responses to Ba(2+), tertiapin-Q, and glibenclamide otherwise evident in diabetic rats. Preglomerular microvascular smooth muscle cells expressed mRNA encoding Kir1.1, Kir2.1, and Kir6.1. Neither diabetes mellitus nor Tempol altered Kir1.1, Kir2.1, Kir6.1, or SUR2B protein levels in renal cortical microvessels. To the extent that the effects of Tempol reflect its antioxidant actions, our observations indicate that oxidative stress contributes to the exaggerated impact of Kir1.1, Kir2.1, and K(ATP) channels on afferent arteriolar tone during diabetes mellitus and that this phenomenon involves posttranslational modulation of channel function.

Nailfold capilloroscopy in systemic lupus erythematosus

IntroductionNailfold capillaroscopy is a non-invasive technique to recognize peripheral microangiopathy, which is an important feature in SLE. Aim of the workTo study the prevalence of nailfold capillaroscopy (NFC) changes in patients with systemic lupus erythematosus (SLE), find out the patterns of these changes and to correlate these findings with different clinical and laboratory parameters. Patients and methodsForty patients with SLE, all fulfilling the 1997 revised criteria for the classification of SLE were included. All patients included in this study were subjected to full history taking, clinical examination, laboratory investigations as well as nailfold capillaroscopy (NFC) examination. ResultsThe prevalence of nailfold capilloroscopic (NFC) changes in SLE patients was 75%. Nailfold capillaroscopic abnormalities were significantly more frequent in SLE patients than in controls (P<0.05). Different abnormal NFC changes were seen with the meandering pattern more frequently seen. The afferent and efferent capillary loops diameters were significantly increased in the SLE patients than in the control group (P<0.05). Some of the NFC abnormalities showed statistical significant correlations with different clinical and laboratory parameters. Capillary loop afferent and efferent diameters were significantly correlated with disease duration (P<0.05) as well as the occurrence of digital gangrene (P<0.05). Also the capillary loop afferent diameter was found to be correlated with the intake of cyclophosphamide (P<0.05). ConclusionSignificant microcirculatory changes occur in systemic lupus erythematosus (SLE) patients as proved by the high prevalence of capillary abnormalities in lupus patients compared to controls by means of nailfold capillaroscopy. Some nailfold capillaroscopy changes e.g. meandering capillaries may complete picture of SLE diagnosis. Duration of SLE disease may have an impact on microcirculation of these patients. The presence of some nailfold capillaroscopy changes in SLE patients may be an alarming sign to fatal ischemia of the digits.

Role of connexin 40 in renal autoregulation

The expression of connexins in renal arterioles is believed to have profound impact on regulation of arteriolar tonus and renal blood flow. Connexin 40 (Cx40), which is primarily expressed in the endothelial cells of the renal afferent arterioles, has been shown to play a significant role in the regulation of renin secretion. Also, connexins are believed to be involved in the transmission of the calcium signal initiating the TGF response and connexins may therefore affect renal autoregulation.We wished to examine the renal autoregulatory response by measuring changes in afferent arteriolar diameter in kidneys from Cx40 knockout (KO) mice in response to a step increase in perfusion pressure. We used the isolated perfused juxtamedullary nephron preparation to visualize afferent arterioles. As previously reported Cx40 KO mice had a higher arterial blood pressure than age‐matched litter‐mate controls. Also, the plasma renin concentration was significantly higher in the Cx40 KO mice compared to wild‐type mice. The change in afferent diameter in response to step increases in renal perfusion pressure was impaired in Cx40 KO mice compared to the wild‐type littermates. These results suggest that Cx40 play a role in the autoregulation of renal blood flow.

High Salt Diet –Induced Afferent Arteriolar Autoregulatory Dysfunction is Improved by Acute Antioxidant Treatment

High salt (HS) diet reduces myogenic reactivity in resistance arteries and autoregulatory responses of afferent arterioles. Oxidative stress is increased in HS rats. We tested the hypothesis that HS blunts autoregulatory behavior by stimulating accumulation of reactive oxygen species. Rats were fed a normal (NS, 0.4% NaCl) or HS (8% NaCl) diet for a period of 14 days. Baseline arteriolar diameter was similar between the NS and HS groups. Autoregulatory behavior was assessed by measuring diameter changes during step‐wise 15 mmHg increases in perfusion pressure from 65 to 170 mmHg. In NS kidneys, afferent arteriolar diameter decreased by 34 ± 2% (n=6) as perfusion pressure increased from 65 to 170 mmHg. In HS kidneys arteriolar diameter decreased by 6 ± 4% (P &lt; 0.05; n = 8) indicating markedly attenuated pressure induced vasoconstriction. Acute exposure to Tempol (1.0 mM) in the superfusate had no effect on the autoregulatory response in NS rats but markedly improved autoregulation in the HS group. Increasing perfusion pressure from 65 to 170 mmHg in NS kidneys decreased afferent diameter by 32 ± 3% (n=3), whereas diameter decreased by 28 ± 3% (n=6) in kidneys from HS rats exposed to Tempol. These data indicate that reactive oxygen species blunt autoregulatory efficiency in kidneys from HS rats facilitating enhanced excretion of salt by buffering autoregulatory control of glomerular hemodynamics.

Rho-kinase inhibition reduces pressure-mediated autoregulatory adjustments in afferent arteriolar diameter.

Preglomerular resistance is regulated by calcium influx- and mobilization-dependent mechanisms; however, the role of Rho-kinase in calcium sensitization in the intact kidney has not been carefully examined. Experiments were performed to test the hypothesis that Rho-kinase inhibition blunts pressure-mediated afferent arteriolar autoregulatory behavior and vasoconstrictor responses evoked by angiotensin II and P2X1 receptor activation. Rat kidneys were studied in vitro using the blood-perfused juxtamedullary nephron technique. Autoregulatory behavior was assessed before and during Rho-kinase inhibition with Y-27632 (1.0 microM; n = 5). Control diameter averaged 14.3 +/- 0.8 microm and increased to 18.1 +/- 0.9 microm (P < 0.05) during Y-27632 treatment. In the continued presence of Y-27632, reducing perfusion pressure to 65 mmHg slightly increased diameter to 18.7 +/- 1.0 microm. Subsequent pressure increases to 130 and 160 mmHg yielded afferent arteriolar diameters of 17.5 +/- 0.8 and 16.6 +/- 0.6 microm (P < 0.05). This 11% decline in diameter is significantly smaller than the 40% decrease obtained in untreated kidneys. The inhibitory effects of Y-27632 on autoregulatory behavior were concentration dependent. Angiotensin II responses were blunted by Y-27632. Angiotensin II (1.0 nM) reduced afferent diameter by 17 +/- 1% in untreated arterioles and by 6 +/- 2% during exposure to Y-27632. The P2X1 receptor agonist, alpha, beta-methylene ATP, reduced afferent arteriolar diameter by 8 +/- 1% but this response was eliminated during exposure to Y-27632. Western blot analysis confirms expression of the Rho-kinase signaling pathway. Thus, Rho-kinase may be important in pressure-mediated autoregulatory adjustments in preglomerular resistance and responsiveness to angiotensin II and autoregulatory P2X1 receptor agonists.

Abstract 1495: Existence of Hyperfiltration in an Early Diabetic Rat was Visualised by Multiphoton Microscopy

To visualise glomerular filtration under physiological conditions in an early diabetic rat and to evaluate filtration molecularly and quantitatively. We used 4 week STZ (50 mg/kg) model as early diabetic rats. We measured diameters of afferent (aff) and efferent (eff) arterioles, glomerular red blood cell velocities and left renal blood flow with our intravital videomicro-scope. Glomerular filtration was visualised by multiphoton microscopy. Various sizes of dextran (3k, 10k, 40k and 70k dalton (Dal)) conjugated with Texas Red in 0.5 ml were administered intravenously by bolus shot and for the next 60 seconds glomerular filtration was observed. FITC conjugated 500k Dal dextran was administered to dye intravascular area. The time course of Texas Red intensity in Bowman’s space was measured from the recorded images. Intensity peak values were normalized by the peak value of 3k Dal as sieving coefficients since the intensity reached its peak within 10 seconds after administration of Texas Red conjugated dextrans. Afferent arteriolar diameters were larger than efferent arteriolar diameters in both control (n=6) and diabetic (n=7) rats. Increases of both afferent and efferent diameters were remarkable in diabetic rats (p&lt;0.05) (aff/eff diameter, DM rats: 14.0 ± 1.9, 9.4 ± 0.7, Control rats: 11.9 ± 0.8, 8.8 ± 0.7 μm). Glomerular red blood cell velocity in diabetic rats was larger than that in control rats (p&lt;0.05) (DM/Control rats: 0.236 ± 0.030, 0.196 ± 0.076 mm/s). Left renal blood flow in diabetic rats was larger than that in control rats (p&lt;0.05) (DM/Control rats: 1.84 ± 0.16, 1.60 ± 0.17 ml/100 gBW). All of these findings suggest the existence of hyperfiltration in early diabetic rats. In fact, filtration of the larger dextrans (40k and 70k Dal) were clearly visualized by multiphoton microscopy in early diabetic rats indicating greater leakage even from the early stage of diabetes with a significant difference by about 20% for the 40k and 70k Dal sieving coefficients comparing with the control group (p&lt;0.05). Both afferent and efferent arteriolar diameters were increased in early diabetic rats suggesting the existence of hyperfiltration and the hyperfiltration was clearly visualised by multiphoton microscopy.

Potassium channel contributions to afferent arteriolar tone in normal and diabetic rat kidney

We previously reported an enhanced tonic dilator impact of ATP-sensitive K+ channels in afferent arterioles of rats with streptozotocin (STZ)-induced diabetes. The present study explored the hypothesis that other types of K+ channel also contribute to afferent arteriolar dilation in STZ rats. The in vitro blood-perfused juxtamedullary nephron technique was utilized to quantify afferent arteriolar lumen diameter responses to K+ channel blockers: 0.1-3.0 mM 4-aminopyridine (4-AP; KV channels), 10-100 microM barium (KIR channels), 1-100 nM tertiapin-Q (TPQ; Kir1.1 and Kir3.x subfamilies of KIR channels), 100 nM apamin (SKCa channels), and 1 mM tetraethylammonium (TEA; BKCa channels). In kidneys from normal rats, 4-AP, TEA, and Ba2+ reduced afferent diameter by 23 +/- 3, 8 +/- 4, and 18 +/- 2%, respectively, at the highest concentrations employed. Neither TPQ nor apamin significantly altered afferent diameter. In arterioles from STZ rats, a constrictor response to TPQ (22 +/- 4% decrease in diameter) emerged, and the response to Ba2+ was exaggerated (28 +/- 5% decrease in diameter). Responses to the other K+ channel blockers were similar to those observed in normal rats. Moreover, exposure to either TPQ or Ba2+ reversed the afferent arteriolar dilation characteristic of STZ rats. Acute surgical papillectomy did not alter the response to TPQ in arterioles from normal or STZ rats. We conclude that 1) KV, KIR, and BKCa channels tonically influence normal afferent arteriolar tone, 2) KIR channels (including Kir1.1 and/or Kir3.x) contribute to the afferent arteriolar dilation during diabetes, and 3) the dilator impact of Kir1.1/Kir3.x channels during diabetes is independent of solute delivery to the macula densa.

Impaired Ca 2+ Signaling Attenuates P2X Receptor–Mediated Vasoconstriction of Afferent Arterioles in Angiotensin II Hypertension

This study tested the hypothesis that afferent arteriolar responses to purinoceptor activation are attenuated, and Ca2+ signaling mechanisms are responsible for the blunted preglomerular vascular reactivity in angiotensin II (Ang II) hypertension. Experiments determined the effects of ATP, the P2X1 agonist beta,gamma-methylene ATP or the P2Y agonist UTP on arteriolar diameter using the juxtamedullary nephron technique and on renal myocyte intracellular Ca2+ concentration ([Ca2+]i) using single cell fluorescence microscopy. Six or 13 days of Ang II infusion significantly attenuated the vasoconstrictor responses to ATP and beta,gamma-methylene ATP (P<0.05). During exposure to ATP (1, 10, and 100 micromol/L), afferent diameter declined by 17+/-2%, 29+/-3%, and 30+/-2% in normal control rats and 8+/-3%, 7+/-3%, and 22+/-3% in kidneys of Ang II-infused rats (13 days). Renal myocyte intracellular calcium responses to ATP or beta,gamma-methylene ATP were also decreased in Ang II hypertensive rats. In myocytes of control rats, peak increases in [Ca2+]i averaged 107+/-21, 170+/-38, and 478+/-79 nmol/L at ATP concentrations of 1, 10, and 100 micromol/L, respectively. Ang II infusion for 13 days decreased the peak responses to ATP (1, 10, and 100 micromol/L) to 65+/-13, 102+/-20, and 367+/-73 nmol/L, respectively. The peak increases in [Ca2+]i in response to beta,gamma-methylene ATP were also reduced in Ang II hypertensive rats. However, angiotensin hypertension did not change the UTP-mediated vasoconstrictor responses or the myocyte calcium responses to UTP. These results indicate that the impaired autoregulatory response observed in Ang II-dependent hypertension can be attributed to impairment of P2X1 receptor-mediated signal transduction.

The CYP450 hydroxylase pathway contributes to P2X receptor-mediated afferent arteriolar vasoconstriction.

This study was conducted to test the hypothesis that the cytochrome P-450 (CYP450) metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) contributes to the afferent arteriolar response to P2 receptor activation. Afferent arteriolar responses to ATP, the P2X agonist, alpha,beta-methylene ATP and the P2Y agonist UTP were determined before and after treatment with the selective CYP450 hydroxylase inhibitor, N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) or the 20-HETE antagonist, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE). Stimulation with 1.0 and 10 microM ATP elicited an initial preglomerular vasoconstriction of 12 +/- 1% and 45 +/- 4% and a sustained vasoconstriction of 11 +/- 1% and 11 +/- 2%, respectively. DDMS or 20-HEDE significantly attenuated the sustained afferent arteriolar constrictor response to ATP. alpha,beta-Methylene ATP (1 microM) induced a rapid initial afferent vasoconstriction of 64 +/- 3%, which partially recovered to a stable diameter 10 +/- 1% smaller than control. Both DDMS and 20-HEDE significantly attenuated the initial vasoconstriction and abolished the sustained vasoconstrictor response to alpha,beta-methylene ATP. UTP decreased afferent diameter by 50 +/- 5% and 20-HEDE did not change this response. In addition, the ATP-induced increase in the intracellular Ca2+ concentration in preglomerular microvascular smooth muscle cells was significantly attenuated by 20-HEDE. Taken together, these results are consistent with the hypothesis that the CYP450 metabolite 20-HETE participates in the afferent arteriolar response to activation of P2X receptors.

Influence of Ca(2+)-activated K(+) channels on rat renal arteriolar responses to depolarizing agonists.

Experiments were performed to evaluate the hypothesis that opening of Ca(2+)-activated K(+) channels (BK(Ca) channels) promotes juxtamedullary arteriolar dilation and curtails constrictor responses to depolarizing agonists. Under baseline conditions, afferent and efferent arteriolar lumen diameters averaged 23.4 +/- 0.9 (n = 36) and 22.8 +/- 1.1 (n = 13) microm, respectively. The synthetic BK(Ca) channel opener NS-1619 evoked concentration-dependent afferent arteriolar dilation. BK(Ca) channel blockade (1 mM tetraethylammonium; TEA) decreased afferent diameter by 15 +/- 3% and prevented the dilator response to 30 microM NS-1619. ANG II (10 nM) decreased afferent arteriolar diameter by 44 +/- 4%, a response that was reduced by 30% during NS-1619 treatment; however, TEA failed to alter afferent constrictor responses to either ANG II or arginine vasopressin. Neither NS-1619 nor TEA altered agonist-induced constriction of the efferent arteriole. Thus, although the BK(Ca) channel agonist was able to curtail afferent (but not efferent) arteriolar constrictor responses to ANG II, BK(Ca) channel blockade did not allow exaggerated agonist-induced arteriolar constriction. These observations suggest that the BK(Ca) channels evident in afferent arteriolar smooth muscle do not provide a prominent physiological brake on agonist-induced constriction under our experimental conditions.

Interactions of adenosine A1 and A2a receptors on renal microvascular reactivity.

Adenosine vasoconstricts preglomerular arterioles via adenosine A1 receptors. Because adenosine also activates adenosine A2 receptors, its overall renal vascular actions are complex and not fully understood. The present study was performed to determine the relative contributions of adenosine A1 and A2a receptors to the responsiveness of the renal microvasculature to adenosine. Afferent and efferent arteriolar diameters were monitored in vitro using the blood-perfused rat juxtamedullary nephron preparation. Basal afferent and efferent arteriolar diameters averaged 17.1 +/- 0.5 (n = 35) and 17.8 +/- 0.5 (n = 20) microm, respectively. Superfusion with 0.1 and 1 micromol/l adenosine did not significantly alter afferent and efferent arteriolar diameters; however, 10 micromol/l adenosine significantly reduced afferent and efferent arteriolar diameters (-8.2 +/- 0.8 and -5.7 +/- 0.6%, respectively). The afferent and efferent arteriolar vasoconstrictor responses to adenosine waned at a dose of 100 micromol/l, such that diameters returned to values not significantly different from control within 2 min. During adenosine A1 receptor blockade with 8-noradamantan-3-yl-1,3-dipropylxanthine (KW-3902: 10 micromol/l), 10 and 100 micromol/l adenosine significantly increased afferent diameter by, respectively, 8.1 +/- 1.2 and 13.7 +/- 1.3% (n = 14) and efferent arteriolar diameter by 6.4 +/- 1.3 and 9.3 +/- 1.2% (n = 8). The afferent and efferent arteriolar vasodilatory responses to adenosine in the presence of KW-3902 were significantly attenuated by addition of the adenosine A2a receptor antagonist 1,3-dipropyl-7-methyl-8-(3,4-dimethoxystyryl)xanthine (KF-17837: 15 micromol/l, n = 7 and 6, respectively). The addition of KF-17837 alone significantly enhanced afferent (n = 15) and efferent (n = 6) arteriolar vasoconstrictor responses to 1, 10, and 100 micromol/l adenosine. These results indicate the presence of adenosine A1 and A2a receptors on afferent and efferent arterioles of juxtamedullary nephrons, such that adenosine A2a receptor-mediated vasodilation partially buffers adenosine-induced vasoconstriction in both pre- and postglomerular segments of the renal microvasculature.

Tyrosine kinase involvement in renal arteriolar constrictor responses to angiotensin II.

Experiments were performed to test the hypothesis that tyrosine kinase activity contributes to renal arteriolar contractile responses to angiotensin (Ang) II. Rats were subjected to short-term enalaprilat treatment to decrease endogenous Ang II formation before tissue was harvested for experiments with the in vitro blood-perfused juxtamedullary nephron technique. Acute surgical papillectomy was used to avoid the indirect afferent arteriolar effect of Ang II that arises through increased tubuloglomerular feedback sensitivity. Arteriolar lumen diameter responses to 1 and 10 nmol/L Ang II were monitored by videomicroscopic methods before and during treatment with various tyrphostin compounds: 100 micromol/L AG18 (broad-spectrum tyrosine kinase inhibitor), 100 nmol/L AG1478 (selective epidermal growth factor receptor tyrosine kinase inhibitor), or 100 micromol/L AG9 (inactive analog). Baseline afferent arteriolar lumen diameter averaged 23.5+/-1.2 micrometer and was not influenced by any tyrphostin. Ang II (10 nmol/L) decreased afferent diameter by 11.1+/-1.0 micrometer under untreated conditions, a response that was not altered by AG9 but significantly blunted by AG18 (34+/-9% inhibition) or AG1478 (52+/-8% inhibition). AG18 did not suppress afferent arteriolar contractile responses to membrane depolarization (20 to 55 mmol/L K(+ )bath). Efferent arteriolar baseline diameter averaged 24.1+/-0.8 micrometer and was unaltered by AG18 or AG1478; however, efferent diameter responses to 10 nmol/L Ang II were diminished 52+/-10% by AG18 and 51+/-13% by AG1478. These observations indicate that Ang II signaling in renal afferent and efferent arteriolar vascular smooth muscle is either mediated or modulated by tyrosine kinase activity, including that of the epidermal growth factor receptor tyrosine kinase.

Contribution of EGF-Receptor Tyrosine Kinase to Angiotensin II-Induced Renal Arteriolar Constriction

P88 Tyrosine kinase activity has been reported to modulate voltage-gated Ca 2+ channels (VGCCs), and has been implicated in both the Ca 2+ mobilization and Ca 2+ influx responses to angiotensin II (AngII). Because renal afferent arteriolar contractile responses to AngII rely on opening of VGCCs while efferent arteriolar AngII responses arise independent of VGCCs, we designed experiments to determine if AngII contracts these arteriolar segments via a mechanism involving tyrosine kinase activation and to examine the role of VGCCs in this phenomenon. Rats were subjected to acute enalaprilat treatment to decrease endogenous AngII formation prior to harvesting tissue for experiments using the in vitro blood-perfused juxtamedullary nephron technique. Papillectomy was employed to avoid the indirect afferent arteriolar effect of AngII that arises via increased tubuloglomerular feedback sensitivity. Arteriolar lumen diameter responses to 1 and 10 nM AngII were monitored using videomicroscopic methods before and during inhibition of EGF-receptor tyrosine kinase activity (100 μM tyrphostin A23). Afferent arteriolar baseline diameter averaged 23.5 ± 1.2 μm ( n = 12) and was unaffected by tyrphostin A23. AngII (10 nM) decreased afferent diameter by 13.7 ± 1.9 μm before and 9.6 ± 1.4 μm during tyrphostin A23 treatment ( n = 7; P + ; n = 5). Neither baseline diameter nor AngII responsiveness was altered by exposure of afferent arterioles to tyrphostin A1 (inactive analog). Efferent arteriolar baseline diameter averaged 23.6 ± 1.1 μm ( n = 6) and was not influenced by tyrphostin A23, but this agent evoked a 52 ± 9% inhibition of the efferent arteriolar diameter response to 10 nM AngII. These observations indicate that 1) EGF-receptor tyrosine kinase activation contributes to AngII-induced contraction of both afferent and efferent arterioles, and 2) a direct influence of the EGF-receptor tyrosine kinase on VGCCs is not likely involved in the afferent arteriolar response to AngII.

Contribution of EGF-Receptor Tyrosine Kinase to Angiotensin II-Induced Renal Arteriolar Constriction

P88 Tyrosine kinase activity has been reported to modulate voltage-gated Ca 2+ channels (VGCCs), and has been implicated in both the Ca 2+ mobilization and Ca 2+ influx responses to angiotensin II (AngII). Because renal afferent arteriolar contractile responses to AngII rely on opening of VGCCs while efferent arteriolar AngII responses arise independent of VGCCs, we designed experiments to determine if AngII contracts these arteriolar segments via a mechanism involving tyrosine kinase activation and to examine the role of VGCCs in this phenomenon. Rats were subjected to acute enalaprilat treatment to decrease endogenous AngII formation prior to harvesting tissue for experiments using the in vitro blood-perfused juxtamedullary nephron technique. Papillectomy was employed to avoid the indirect afferent arteriolar effect of AngII that arises via increased tubuloglomerular feedback sensitivity. Arteriolar lumen diameter responses to 1 and 10 nM AngII were monitored using videomicroscopic methods before and during inhibition of EGF-receptor tyrosine kinase activity (100 μM tyrphostin A23). Afferent arteriolar baseline diameter averaged 23.5 ± 1.2 μm ( n = 12) and was unaffected by tyrphostin A23. AngII (10 nM) decreased afferent diameter by 13.7 ± 1.9 μm before and 9.6 ± 1.4 μm during tyrphostin A23 treatment ( n = 7; P &lt; 0.05), indicating 28 ± 8% inhibition of AngII-induced constriction. However, tyrphostin A23 did not suppress afferent arteriolar contractile responses to membrane depolarization (20-55 mM bath K + ; n = 5). Neither baseline diameter nor AngII responsiveness was altered by exposure of afferent arterioles to tyrphostin A1 (inactive analog). Efferent arteriolar baseline diameter averaged 23.6 ± 1.1 μm ( n = 6) and was not influenced by tyrphostin A23, but this agent evoked a 52 ± 9% inhibition of the efferent arteriolar diameter response to 10 nM AngII. These observations indicate that 1) EGF-receptor tyrosine kinase activation contributes to AngII-induced contraction of both afferent and efferent arterioles, and 2) a direct influence of the EGF-receptor tyrosine kinase on VGCCs is not likely involved in the afferent arteriolar response to AngII.

Effects of angiotensin II on regional afferent and efferent arteriole dimensions and the glomerular pole.

The diversity of renal arteriole diameters in different cortical regions has important consequences for control of glomerular capillary pressure. We examined whether intrarenal angiotensin II (ANG II; 0.1, 1, or 5 ng. kg(-1). min(-1)) in anesthetized rabbits acts preferentially on pre- or postglomerular vessels using vascular casting. ANG II produced dose-related reductions in afferent and efferent diameters in the outer, mid, and inner cortex, without effecting arterial pressure. Afferent diameter decreased more than efferent in the outer and mid cortex (P < 0.05) but by a similar extent in juxtamedullary nephrons (P = 0.58). Calculated efferent resistance increased more than afferent, especially in the outer cortex (127 vs. 24 units; 5 ng. kg(-1). min(-1) ANG II). ANG II produced significant dose-related increases in the distance between the arterioles at the entrance to the glomerular pole in all regions. Thus afferent diameter decreased more in response to ANG II, but efferent resistance rose more due to smaller resting luminal dimensions. The results also indicate that glomerular pole dimensions change in response to ANG II.

Renal arteriolar contractile responses to angiotensin II in rats with poorly controlled diabetes mellitus.

1. Experiments were performed to test the hypothesis that renal arteriolar responses to angiotensin (Ang)II are altered in insulin-dependent diabetes mellitus. 2. Male Sprague-Dawley rats were treated with streptozotocin (STZ; 65 mg/kg, i.v.) or 0.9% NaCl vehicle (Sham). Partial insulin replacement maintained blood glucose levels at 422 +/- 6 mg/dL (STZ rats) for the ensuing 2 week period (86 +/- 4 mg/dL in Sham rats). The in vitro blood-perfused juxtamedullary nephron technique was used to study renal arteriolar diameter responses to exogenous AngII or K(+)-induced membrane depolarization. 3. Baseline afferent arteriolar diameter did not differ between kidneys harvested from Sham and STZ rats; however, constrictor responses to AngII were accentuated in kidneys from diabetic rats. During exposure to 10 nmol/L AngII, afferent diameter decreased by 37 +/- 5 and 18 +/- 4% in STZ and Sham kidneys, respectively. Efferent arterioles from Sham and STZ rats did not differ with regard to either baseline diameter or AngII responsiveness. 4. In experiments assessing afferent arteriolar responsiveness to membrane depolarization, 40 mmol/L K+ decreased lumen diameter by 73 +/- 11% in kidneys from Sham rats; however, the same depolarizing stimulus only reduced afferent diameter by 28 +/- 7% in STZ kidneys. 5. We conclude that afferent (but not efferent) arteriolar AngII responsiveness is increased during the early stages of poorly controlled diabetes mellitus in the rat. The exaggerated afferent arteriolar responsiveness to AngII occurs despite reduced sensitivity to membrane depolarization, suggesting that the emergence of alternative signalling processes or alterations in vasoactive modulator influences may underlie this phenomenon.

Suppressed impact of nitric oxide on renal arteriolar function in rats with chronic heart failure.

We performed experiments to test the hypothesis that experimental heart failure (HF) is associated with altered nitric oxide (NO)-dependent influences on the renal microvasculature, including diminished modulation of constrictor responses to ANG II. Eight to ten weeks after inducing HF in rats by coronary artery ligation, we administered enalaprilat to suppress ANG II synthesis and studied renal arteriolar function using the in vitro blood-perfused juxtamedullary nephron technique. In kidneys from sham-operated rats, NO synthase inhibition [100 microM Nomega-nitro-L-arginine (L-NNA)] reduced afferent arteriolar diameter by 4.1 +/- 0.6 microm and enhanced ANG II responsiveness (10 nM ANG II decreased afferent diameter by 10.1 +/- 1.4 micrometer before and 12.8 +/- 1.6 micrometer during L-NNA treatment; P < 0.05). In kidneys from HF rats, L-NNA did not alter afferent arteriolar baseline diameter or ANG II responsiveness (10 nM ANG II decreased diameter by 12.5 +/- 1.5 micrometer before and 12.5 +/- 2.3 micrometer during L-NNA). The effects of L-NNA on efferent arteriolar function were also abated in HF rats. In renal cortex of HF rats, NO synthase activity was decreased by 63% and superoxide dismutase activity was diminished by 39% relative to tissue from sham-operated rats. Urinary nitrate/nitrite excretion was also reduced in HF rats. Thus both diminished synthesis and augmented degradation are likely to contribute to a decreased renal microvascular impact of endogenous NO during chronic HF, the consequences of which include loss of NO-dependent modulation of ANG II-induced vasoconstriction.