Abstract P247: Elastin Insufficiency Impairs Renal Hemodynamics And Accelerates Aging-associated Structural And Functional Remodeling Of Preglomerular Arterioles

Elastin degradation and fragmentation are hallmarks of arterial stiffness and renal dysfunction associated with aging. However, it is unclear whether elastin insufficiency contributes to the changes in the structure and function of the resistance vasculature of the kidney during aging. Here we determined how increased vascular stiffness due to elastin insufficiency alters renal hemodynamics and mechanical properties of preglomerular arterioles. We assessed renal hemodynamics under anesthesia in 14-16-month-old female wild type (WT) and elastin heterozygous ( Eln +/- ) mice. Renal autoregulation was assessed by a stepwise increase in renal perfusion pressure (RPP) by simultaneously occluding the superior mesenteric and celiac arteries. Myogenic constriction and arterial stiffness were assessed by pressure myography of isolated renal interlobar arteries. Baseline renal vascular resistance (RVR) was elevated in Eln +/- mice (13.8 ± 2.9 vs 11.2 ± 1.4 mmHg/μL/min/g left kidney weight), while systolic blood pressure (SBP; 75.1 ± 7.4 vs 91 ± 4.2mmHg), renal blood flow (RBF; 6.3 ± 1.2 vs 7.4 ± 1.7 μL/min/g left kidney weight), renal plasma flow (RPF; 3.4 ± 0.8 vs 5 ± 1.2 mL/min/g/ left kidney weight) and urine flow rate, all trended lower in Eln +/- mice compared to WT mice. Glomerular filtration rate (GFR) and filtration fraction (FF) were similar between the two groups. A stepwise increase in RPP caused a slower decline and rise in RBF and RVR, respectively, in Eln +/- relative to WT mice. The maximal changes in RBF (5 ± 1.1 vs 4.7 ± 0.8 μL/min/g left kidney weight), RVR (17.6 ± 7.3 vs 22.5 ± 2.1 mmHg/μL/min/g left kidney weight), urine flow rate,GFR, and FF were less robust in Eln +/- mice. RPF decreased in WT mice in response to raising RPP, whereas it remained unchanged in Eln +/- mice. Myogenic response and increases in elastic modulus and wall tension following stepwise changes in intraluminal pressure were all augmented in interlobar arteries from Eln +/- relative to WT mice. However, there was no difference in kidney weight/tibia length ratio between the two genotypes. We conclude that elastin insufficiency impairs renal hemodynamics by exacerbating age associated increase in vascular stiffness.

Connexin mimetic peptides fail to inhibit vascular conducted calcium responses in renal arterioles

Vascular conducted responses are believed to play a central role in controlling the microcirculatory blood flow. The responses most likely spread through gap junctions in the vascular wall. At present, four different connexins (Cx) have been detected in the renal vasculature, but their role in transmission of conducted vasoconstrictor signals in the preglomerular arterioles is unknown. Connexin mimetic peptides were previously reported to target and inhibit specific connexins. We, therefore, investigated whether conducted vasoconstriction in isolated renal arterioles could be blocked by the use of mimetic peptides directed against one or more connexins. Preglomerular resistance vessels were microdissected from kidneys of Sprague-Dawley rats and loaded with fura 2. The vessels were stimulated locally by applying electrical current through a micropipette, and the conducted calcium response was measured 500 μm from the site of stimulation. Application of connexin mimetic peptides directed against Cx40, 37/43, 45, or a cocktail with equimolar amounts of each, did not inhibit the propagated response, whereas the nonselective gap junction uncoupler carbenoxolone completely abolished the propagated response. However, the connexin mimetic peptides were able to reduce dye coupling between rat aorta endothelial cells shown to express primarily Cx40. In conclusion, we did not observe any attenuating effects on conducted calcium responses in isolated rat interlobular arteries when exposed to connexin mimetic peptides directed against Cx40, 37/43, or 45. Further studies are needed to determine whether conducted vasoconstriction is mediated via previously undescribed pathways.

Interactions of adenosine A1 and A2areceptors on renal microvascular reactivity

Adenosine vasoconstricts preglomerular arterioles via adenosine A1receptors. Because adenosine also activates adenosine A2receptors, 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) μm, respectively. Superfusion with 0.1 and 1 μmol/l adenosine did not significantly alter afferent and efferent arteriolar diameters; however, 10 μmol/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 μmol/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 μmol/l), 10 and 100 μmol/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 μmol/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 μmol/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.

Mechanisms Mediating the Renal Profibrotic Actions of Vasoactive Peptides in Transgenic Mice

Transgenic mice are useful tools to investigate the mechanisms of the renal profibrotic actions of endothelin and angiotensin II. The overexpression of angiotensinogen and renin genes induces renal sclerosis independently of changes in systemic hemodynamics. The same results are observed when the endothelin-1 gene is overexpressed. Transgenic mice harboring the luciferase gene, under the control of the collagen I α2 chain promoter, and made hypertensive by induction of a nitric oxide (NO) deficiency have been studied. In this strain of mice, luciferase activity is an early index of renal and vascular fibrosis. Luciferase activity was increased in preglomerular arterioles and glomeruli when mice were treated with Nω-nitro-L-arginine methyl ester, an inhibitor of NO synthases. Bosentan (an endothelin receptor antagonist) was as efficient as losartan (an AT1 receptor antagonist) in preventing renal fibrosis, although it did not decrease BP. In short-term experiments, angiotensin II produced an increase in luciferase activity that was entirely prevented by losartan but also by bosentan. It can be concluded that, during chronic inhibition of NO, the collagen I gene is activated, which contributes to the development of nephroangiosclerosis and glomerulosclerosis. Angiotensin II plays a major role in this fibrogenic process, and its effect is at least partly independent of systemic hemodynamics and mediated by the profibrotic action of endothelin-1.

Upregulation of V1 receptors in renal resistance vessels of rats developing genetic hypertension

Previous studies have demonstrated that arginine vasopressin (AVP) produces exaggerated renal vasoconstriction in young spontaneously hypertensive rats (SHR) relative to normotensive rats. The exaggerated renal vascular reactivity does not appear to be due to a primary defect in postreceptor calcium signal transduction. Although the magnitudes of vascular responses differ, the relative proportions of calcium entry and mobilization pathways evoked by AVP in renal resistance vessels are similar in these rat strains. The purpose of the present study was to evaluate possible differences in V1 mRNA and receptor density and affinity in preglomerular resistance vessels (<50 μm) obtained from young Wistar-Kyoto (WKY) and SHR. Quantitative RT-PCR analysis revealed twofold greater expression of the V1areceptor gene in preglomerular arterioles of 7-wk-old SHR compared with WKY. In vitro radiolabeled ligand binding studies were performed under equilibrium conditions on preglomerular resistance arterioles freshly isolated from kidneys of 7-wk-old rats. The results indicate that AVP receptor density (Bmax) is two to three times greater in SHR than in WKY (248 ± 24 vs. 91 ± 11 fmol/mg protein, P < 0.001). The affinity does not differ between strains ( K d = 0.5 nM). Displacement studies yielded similar results for SHR and WKY. Unlabeled AVP completely displaced [3H]AVP binding, with an IC50 of 2.5 × 10− 10 M. Expression of AVP receptor types in afferent arterioles was evaluated using the V1 receptor agonist, [Phe2, Ile3,Org8]vasopressin, the V1 receptor antagonist, [d(CH2)5, Tyr(Me)2,Tyr(NH2)9]Arg8-vasopressin, and the V2 receptor agonist, desamino-[d-Arg8]vasopressin. Both the V1 agonist and antagonist displaced up to 90% of the AVP binding with IC50 values of 4 × 10− 8 and 8 × 10− 7 M, respectively. The V2receptor agonist was a weak inhibitor, displacing less than 15% of AVP binding at a high concentration of 10− 4M. These results demonstrate that virtually all AVP receptors in the preglomerular arterioles are of the V1 type. Collectively, our results provide evidence that the enhanced renal reactivity to AVP is mediated by a higher density of V1 receptors associated with increased gene expression in renal resistance vessels of SHR developing genetic hypertension.

Prostaglandins buffer ANG II-mediated increases in cytosolic calcium in preglomerular VSMC

In order to exert an appropriate biological effect, the action of the vasoconstrictive hormone angiotensin II (ANG II) is modulated by vasoactive factors such as prostaglandins PGE2 and PGI2. The present study investigates whether prostaglandins alter ANG II-mediated increases in cytosolic calcium concentration ([Ca2+]i) in vascular smooth muscle cells (VSMC) isolated from rat renal preglomerular arterioles. [Ca2+]i was assessed using the calcium-sensitive dye fura 2 and a microscope-based photometer system. ANG II (10−7 M) caused a biphasic, time-dependent [Ca2+]i response: an initial peak increase from 52 ± 7 to 264 ± 25 nM, followed by a sustained plateau of 95 ± 9 nM in cultured VSMC. Coadministration of PGE2 or PGI2 or synthetic mimetics caused dose-dependent decreases in the peak [Ca2+]i response to ANG II, with attenuation of 40–50%. This degree of inhibition was even more pronounced in individual freshly isolated preglomerular VSMC. Increasing cAMP levels in cultured VSMC, by using either a cell-permeable analog or inhibiting phosphodiesterase activity, mirrored the antagonistic effects of prostaglandins on ANG II-stimulated increases in [Ca2+]i. Radioimmunoassays demonstrate that ANG II (10−7 M) stimulates production of PGI2 and PGE2; the stable prostacyclin metabolite 6-keto-PGF1 αwas released in 10-fold greater concentrations than PGE2.Indomethacin blockade of prostaglandin production potentiated both the peak (264 to 337 ± 26 nM) and sustained [Ca2+]i responses (95 to 181 ± 22 nM) to ANG II. When prostaglandin analogs were added during indomethacin treatment, the ANG II response was restored to the typical pattern. In conclusion, we demonstrate that modulation of intracellular calcium levels is one mechanism by which prostaglandins can buffer ANG II-mediated constriction in renal preglomerular VSMC. PGI2 is more potent than PGE2 in this regard.

20-HETE and the kidney: resolution of old problems and new beginnings

The protean properties of 20-hydroxyeicosatetraenoic acid (HETE), vasoactivity, mitogenicity, and modulation of transport in key nephron segments, serve as the basis for the essential roles of 20-HETE in the regulation of the renal circulation and electrolyte excretion and as a second messenger for endothelin-1 and mediator of selective renal effects of ANG II. Renal autoregulation and tubular glomerular feedback are mediated by 20-HETE through constriction of preglomerular arterioles, responses that are maintained by 20-HETE inhibition of calcium-activated potassium channels. 20-HETE modulates ion transport in the proximal tubules and the thick ascending limb by affecting the activities of Na+-K+-ATPase and the Na+-K+-2Cl−cotransporter, respectively. The range and diversity of activity of 20-HETE derives in large measure from COX-dependent transformation of 20-HETE to products affecting vasomotion and salt and water excretion. Nitric oxide (NO) exerts a negative modulatory effect on 20-HETE formation; inhibition of NO synthesis produces marked perturbation of renal function resulting from increased 20-HETE production. 20-HETE is an essential component of interactions involving several hormonal systems that have central roles in blood pressure homeostasis, including angiotensins, endothelins, NO, and cytokines. 20-HETE is the preeminent renal eicosanoid, overshadowing PGE2 and PGI2. This review is intended to provide evidence for the physiological roles for cytochrome P-450-derived eicosanoids, particularly 20-HETE, and seeks to extend this knowledge to a conceptual framework for overall cardiovascular function.