Control of Renal Cortical Microvascular Resistance

Methods of Evaluation

Much of the recent focus on cortical microcirculation research has been on identification of the systems that regulate vascular tone, affecting segmental and overall renal vascular resistance. Access to the important resistance vessels is limited, but recent methodological gains have provided new information. Both in vivo and in vitro observations continue to be made in hydronephrotic kidneys, developed by prolonged ureteral occlusion. However, there is concern that responses in this nonfiltering kidney model are not fully representative of normal function. The recent use of intravital microscopy, with improved optics and imaging analysis, shows promising results suggesting that in vivo inspection of renal cortical microcirculation in normal kidneys is possible. Isolated segments of the interlo-bar, arcuate, and interlobular arteries continue to provide data, though they often are similar to nonrenal vessels. However, more studies have emerged from direct perfusion of the isolated afferent arterioles and in some cases the efferent arterioles, which provide more renal specific responses. These extremely small (15 to 30 mm) vessels have been isolated from rabbits, mice and more recently rats and micro-perfused in temperature-controlled baths. Though many of these observations may be limited to the experimental species, they provide a sensitive resistance vessel model with which to test several hypotheses. Renal micropuncture also continues to provide data on the control of cortical vascular tone.

Angiotensin II

Angiotensin II is the major end product of the proteolytic enzyme renin, which is primarily produced in the kidney in the afferent arteriole. Ang II is a potent vasoconstrictor in vascular beds mediated by abundant type 1 (AT1) receptors. Type 2 receptors (AT2), which are much less abundant, may cause vasodilation when activated, but supporting evidence for a physiological role is currently lacking. Ang II may have unique vascular control in the kidney, since it is well established that the level of Ang II-dependent constriction varies between afferent and efferent arterioles. Several new studies have focused on the relative contributions of Ang Il-dependent constriction of the AA and EA. In an attempt to characterize Ang I and Ang II actions in these arterioles, Marchetti et al. [1] measured changes in intracellular Ca2+ (Caj) concentrations as a parameter of vasoactive responses in isolated rat juxtaglomerular arterioles. They studied three populations of arterioles isolated from juxtamedullary glomeruli, the afferent arterioles (AA), efferent arterioles terminating as peritubular capillaries (EA-T), and EA terminating as vasa recta (EA-M). Cai was increased in all arterioles by both Ang I and II and was blocked in all arterioles by losartan, an Ang II receptor blocker. Lisinopril (an angiotensin-converting enzyme inhibitor) blocked the increase in Cai due to Ang I in the AA and the EA-M, yet had no effect in EA-T. The ratio of EC50 values for Ang I to Ang II was also much higher in EA-M. The authors suggest that local ACE acts to convert Ang I to Ang II, which then activates AT receptors and initiates Cai mobilization and constriction. However, in the EA-M, which may have no ACE activity, Ang I either activates the Ang II receptors or is converted to some other active form. The authors fail to identify a physiological role for this arteriolar heterogeneity. The same group also assessed expression of AT1a, AT1b, and AT2 receptors in these microdissected arterioles. All three receptors were expressed in AA and EA-M. Yet, only modest levels of mRNA for AT1a and AT2 were expressed in EAT. The expression of the receptor subtypes correlates with the Ang II effects on Ca2+ mobilization.

The arteriolar diameter responses to systemic infusion of Ang II were also evaluated in the dog kidney using intravital microscopy [2]. This improved imaging method may provide direct evidence of differential responses to Ang II. However, renal artery injections of Ang II reduced diameters in EA and AA similarly in superficial glomeruli. Total constriction differed only modestly between the AA and EA in the juxtamedullary glomeruli. Inhibition of prostaglandins enhanced the Ang II response only in the jux-tamedullary AA, suggesting that PG vasodilating products offset Ang II in the juxtamedullary AA, but not in the cortical AA. However, nitric oxide may also contribute to the Ang II response in juxtamedullary AA. This study was able to demonstrate zonal heterogeneity of Ang II control of vascular tone. The lack of sufficient resolution made it difficult to distinguish major differences between AA and EA responses to Ang II, which has been demonstrated more clearly in isolated perfused arterioles. Another weakness of this technique is the inability to inject vasoactive agents directly into the microvasculature in the video field to ascertain local responses.

Adenosine Receptors

Adenosine, acting on type 1 receptors (A1-R) in the afferent arteriole, constricts the AA, reduces the GFR, and also mediates tubuloglomerular feedback (TGF). Local adenosine levels are increased during elevated Na+ transport in the nephron and subsequent metabolism of adenosine triphosphate (ATP). The relationship between Ang II and adenosine on the regulation of afferent arteriolar tone has been the focus of several studies. These agents act through AT1 and A1 receptors that have been localized in the AA. Both acute and chronic suppression of the renin-angiotensin system (RAS) reduces A1-R vasoconstriction. The dependence of Ang II on A1-R is not as clear. In the hydronephrotic kidney, A1-R inhibition had no effect on Ang II constriction. However, in isolated perfused rabbit AA, A1-R inhibition reduced Ang II constriction by 50 percent. With the development of A1-R knockout mice, this question was re-addressed to test the chronic effects of A1-R inhibition. Hansen et al. [3] measured renal function in anesthetized mice and in separate experiments tested the tone of the isolated perfused AA in response to Ang II. The GFR did not differ between wild type and mice lacking A1-R during the control period. An acute pressor dose of Ang II raised MAP similarly in both groups (+11 to +13 mmHg). However, Ang II reduced GFR by 40 to 50 percent in the wild-type (+/+) mice, but only by 20 percent in the A1-R (-/-) mice (Figure 1). Ang II reduced the renal blood flow (RBF) more in the knockout mice, similar to its effect on GFR. In the micro-perfused AA the baseline diameters were similar between the groups. However, Ang II was more effective in the A1-R (+/+) than in the A1-R (-/-) in reducing AA diameter. At a physiological dose (10-10M) Ang II reduced the diameter in A1-R (+/+) by 50 percent, yet had no effect in A1-R (-/-). These differences were probably not due to differences in AT1 receptor density, since the expression of mRNA for ATj was not different between AA from the two

Figure 1 Glomerular filtration rate (GFR) of wild-type and A1 adenosine receptor (A1R) knockout mice in three consecutive 10-minute periods during control and during intravenous infusion of Ang II at 1.5 ng/min. Values are means of 6 experiments SE. *Significance between A1R +/+ and -/- for a given period (p < 0.05).

Figure 1 Glomerular filtration rate (GFR) of wild-type and A1 adenosine receptor (A1R) knockout mice in three consecutive 10-minute periods during control and during intravenous infusion of Ang II at 1.5 ng/min. Values are means of 6 experiments SE. *Significance between A1R +/+ and -/- for a given period (p < 0.05).

groups. This study provides clear evidence that the full effect of Ang II constriction in the AA requires a functioning A1-R, and presumably activation by increased adenosine release. Alternately, the level of interaction could be mediated by G-protein coupled signaling of these two receptors.

Purinergic Receptors

The vascular control mediated by adenosine and its A1-R may be closely related to ATP-linked purinergic receptors, particularly in the renal cortical circulation where both families of receptors are abundantly expressed. The P2X (ATP) and P2Y (UTP) families of receptors, specifically P2Xb P2X3, and P2Y2, mediate increases in intracellular calcium concentration in vascular smooth muscle cells and perhaps vascular tone in the afferent arterioles. Activation of both types of receptors in freshly harvested preglomerular smooth muscle cells increases intracellular calcium concentration, presumably through different mechanisms. To test this hypothesis in a vascular preparation, Inscho and Cook [4] measured the diameters of rat juxtamedullary AA in response to perfusion of ATP and UTP with and without treatment of diltiazem, a calcium channel blocker. The P2 agonists a,P-methylene ATP, ATP, and UTP reduced the diameters of AA by 8 to 30 percent (Figure 2). The constrictive response to the specific P2Xj agonist a,b-methyl-ene ATP was completely blocked by diltiazem, which suggests that this receptor acts to increase calcium concentration from extracellular sources. The constrictive response to ATP at doses less than 10 ||M was also blocked by diltiazem. However, the response to UTP was similar before and during diltiazem. These physiological observations confirm previous studies in cells that ATP and UTP increase intra-cellular calcium, which is consistent with the increased tone of the AA elicited in this study. Further, the increases to ATP appear to be mediated via L-type channels from extracellular sources and the increases to UTP from release of intracellular stores.

Arachidonic Metabolites

The major product of cyclooxygenase activity in the kidney is prostaglandin E2 (PGE2), which mediates vasodilation through activation of the EP2 receptor. Four G protein-coupled EP receptors have been identified in the kidney, and the physiological effects of these have not been fully established. In an attempt to clarify these roles, Imig et al. [5] tested the effects of PGE2 in mice with disrupted EP2 receptors. They used a preparation in the isolated perfused mouse kidney that surgically exposes the juxtamedullary glomeruli. PGE2 added to the perfusate dilated the precon-stricted afferent arteriole in EP2 (+/+) mice, but further constricted the AA in EP2 (-/-) mice (Figure 3). This suggests that the renal vasodilation associated with PGE2 is mediated specifically by the EP2 receptor. In the absence of EP2, PGE2 activates other EP receptors, which causes vasoconstriction. Selective inhibition of EPj and EP3 receptors prevented the PGE2-associated constriction. In addition this constriction was also blocked with ACE inhibition, demonstrating the link between PGE2 and Ang II. EP2 receptors may be critically involved in maintenance of renal blood flow or glomerular filtration, specifically by mediating the vasodila-tion associated with PGE2.

Endothelially Derived Hormones

The relative resistance of preglomerular arteries and the afferent arteriole is often considered when assessing total renal vascular resistance and its hormonal control. Larger arteries are more accessible and are often used for isolated in vitro studies, yet the well-established dominant resistance

Figure 2 Afferent arteriolar responses to repeat applications of P2 agonists. *Significant reduction in diameter compared with the preceding control diameter (i < 0.05).

Figure 3 Afferent arteriolar diameter responses to PGE2 in EP2 +/+ and EP2 —/— mice. The afferent arteriolar PGE2 responses in EP2 +/+ mice are shown in A, and PGE2 —/— mice are shown in B. Diameter measurements at 15-second intervals are depicted under control conditions (first 5 minutes) and after addition of PGE2 (second 5 minutes).

Figure 3 Afferent arteriolar diameter responses to PGE2 in EP2 +/+ and EP2 —/— mice. The afferent arteriolar PGE2 responses in EP2 +/+ mice are shown in A, and PGE2 —/— mice are shown in B. Diameter measurements at 15-second intervals are depicted under control conditions (first 5 minutes) and after addition of PGE2 (second 5 minutes).

capacity of the afferent arteriole makes this tissue more applicable in control of glomerular hydrostatic pressure and RBF. However, because of the small size and fragility of this tissue, there have been only a few in vitro observations in this key resistance vessel, and these have been limited to rabbits and mice. Several hormones have been implicated in the control of larger artery resistance in studies using isolated preglomerular vessels. Wang et al. [6] recently evaluated several vasodilators in the isolated perfused rabbit AA. Nitric oxide (NO) and endothelium-derived hyperpolarizing factors (EDHF) mediate acetylcholine-induced vasodilation in small vessels, and the relative effects of these have been fully investigated in the larger preglomerular arteries. EDHF may be more important in smaller resistance vessels, yet the relative roles have not been studied extensively in the AA. The epoxide product epoxyeicosatrienoic acid (EET) may mediate vasodilation in these vessels, but another epoxide 20-hydroxyeicosatetraenoic acid (20-HETE) acts as a vasoconstrictor and may offset its effect. Wang et al. tested the roles of EDHF, NO, and prostaglandins on the Ach-induced dilation in isolated preconstricted AA [6]. COX inhibition had no effect on the Ach-induced vasodilation, whereas more than 50 percent could be blocked with inhibition of NO. Most of the remaining dilation was blocked by inhibitors of Ca2+ activated K+ channels or by high extracellular K+, which suggests EDHF. In an effort to characterize the EDHF, the tissue was treated with a competitive inhibitor of 14,15-EET, the vasodilating epoxide identified in other renal arteries. During NO inhibition 14,15-epoxye-icosa-5(Z)-enoic acid (14,15-EEZE) blocked much of the remaining dilation, equal to K+ channel blockade. Both inhibitors, however, did not account for all of the Ach-induced vasodilation in this model. Though this study iden tified a novel dilator in the AA, the level of NO-dependent dilation was greater than in studies with other small-resistance vessels.

Rho/Rho Kinase

More novel pathways that may regulate renal vascular tone continue to be described and investigated. In vascular smooth muscle cells (VSMC), one of the final steps for contraction is the increased intracellular Ca2+ activation of myosin light chain (MLC) kinase, which leads to MLC phosphorylation (MLCP). However, several studies have shown that MLCP inhibition increases Ca2+ sensitivity and contraction in VSMC. One endogenous mechanism that checks MLCP is the activation of Rho kinase (ROK) by the G protein RhoA. Recently developed ROK inhibitors have been used to characterize its role in constriction. The early use of these compounds reduced in vitro vascular contraction, which is consistent with the theoretical control of ROK on Ca2+ sensitivity. However, since ROK increases stress fiber formation and actin polymerization, the vasoconstriction could occur via these mechanisms. Cavarape et al. [7] were the first to test the effects of ROK inhibitors on the renal vascular tone. In the in vivo hydronephrotic rat kidney, they showed that ROK inhibition increased glomerular blood flow and dilated interlobular arteries, and afferent arterioles more than efferent arterioles. They further tested agonists to three small renal artery constrictors that all increase intercellular Ca2+: endothelin B receptors (ETB), adenosine 1 receptors (A1-R), and guanylyl cyclase (GC). ETB and A1-R are more effective on AA and GC is more effective in EA. ROK inhibitors prevented the constriction associated with each agonist in nearly all vessels. ROK

inhibitors had no effect on expression of actin; therefore the authors conclude that the action of ROK inhibition is on renal VSMC Ca2+ sensitivity.

Ischemia

The recent development of miniaturized video imaging and improved image analysis has spurred the study of cortical microcirculation under normal and experimental conditions. With a pencil-like lens of 1 mm diameter, this video system is capable of visualizing the glomerular and pre- and postglomerular circulation, as well as interlobular arteries. This improved instrument has been validated by confirming microcirculatory responses to Ang II and TGF stimulation originally observed by in vivo renal micropuncture. Early use of this instrument was made in anesthetized dogs, but now applications are made in the rat, as well. Evaluations of microcirculatory changes due to pathophysiology and responses to vasoactive agents are now emerging.

In a study that demonstrates the capabilities of this new tool, Yamamoto et al. [8] observed peritubular capillaries, glomerular arterioles and interlobular arteries before and after 45 minutes of renal artery occlusion in the rat (Figure 4). By measuring the velocity of red blood cells (RBC), they showed that blood flow quickly returned to normal levels during the first 1 to 2 minutes after release of the occlusion. However, capillary flow dropped to levels 10 to 20 percent of normal during the first 2 to 5 minutes after release and gradually improved over the next 60 minutes (Figure 5). Similar observations were made in the glomerular blood flow, except that flow was fully restored after 60 minutes. This could not be explained by interlobular artery flow, since these vessels tended to dilate after occlusion. A second finding was that flow through the peritubular capillaries was always orthograde during the control period, but after ischemia the authors noted a mixture of orthograde, retrograde and no flow. The lack of perfusion of all peritubular capillaries confirms and strengthens the "no-flow" theory of postischemic vascular damage, directly demonstrating profound and sustained reduction of flow. The authors conclude that 45 minutes of renal ischemia can induce vascular nephropathy.

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