Pallone, Thomas L., Zhong Zhang, and Kristie Rhinehart. Physiology of the renal medullary microcirculation. Am J Physiol Renal Physiol 284: F253-F266, 2003; 10.1152/ajprenal.00304.2002.-Perfusion of the renal medulla plays an important role in salt and water balance. Pericytes are smooth muscle-like cells that impart contractile function to descending vasa recta (DVR), the arteriolar segments that supply the medulla with blood flow. DVR contraction by ANG II is mediated by depolarization resulting from an increase in plasma membrane Cl Ϫ conductance that secondarily gates voltage-activated Ca 2ϩ entry. In this respect, DVR may differ from other parts of the efferent microcirculation of the kidney. Elevation of extracellular K ϩ constricts DVR to a lesser degree than ANG II or endothelin-1, implying that other events, in addition to membrane depolarization, are needed to maximize vasoconstriction. DVR endothelial cytoplasmic Ca 2ϩ is increased by bradykinin, a response that is inhibited by ANG II. ANG II inhibition of endothelial Ca 2ϩ signaling might serve to regulate the site of origin of vasodilatory paracrine agents generated in the vicinity of outer medullary vascular bundles. In the hydropenic kidney, DVR plasma equilibrates with the interstitium both by diffusion and through water efflux across aquaporin-1. That process is predicted to optimize urinary concentration by lowering blood flow to the inner medulla. To optimize urea trapping, DVR endothelia express the UT-B facilitated urea transporter. These and other features show that vasa recta have physiological mechanisms specific to their role in the renal medulla. vasa recta; perfusion; hypertension; oxygenation; urinary concentration; patch clamp; calcium; fura 2 THE MICROCIRCULATION OF THE kidney is regionally specialized. In the cortex, afferent and efferent arterioles govern the driving forces that promote glomerular filtration. A dense peritubular capillary plexus arising from efferent arterioles surrounds the proximal and distal convoluted tubules to accommodate enormous reabsorption of glomerular filtrate. In contrast, vasa recta serve needs specific to the medulla. Through the counterflow arrangement of descending (DVR) and ascending vasa recta (AVR), countercurrent exchange traps NaCl and urea deposited to the interstitium by collecting ducts and the loops of Henle. This is vital to maintain corticomedullary osmotic gradients but conflicts with the need to supply nutrient blood flow to medullary tissue. Metabolic substrates that enter the medulla in DVR blood diffuse to the AVR to be shunted back to the cortex. To deal with the threat of medullary hypoxia resulting from this process, the kidney has evolved a capacity to exert subtle control over regional perfusion of the outer and inner medulla. The details are far from clear, but much experimental evidence points to the complex interactions of many autocoids and paracrine agents to modulate vasomotor tone at various sites along the microvascular circuit. The goal of this review is to summarize ...

We devised a method for removal of pericytes from isolated descending vasa recta (DVR). After enzymatic digestion, aspiration of a descending vas rectum into a micropipette strips the pericytes from the abluminal surface. Pericytes and denuded endothelia can be recovered for separate study. Using fura 2-loaded preparations, we demonstrated that 10 nM angiotensin II (ANG II) elevates pericyte intracellular Ca(2+) concentration ([Ca(2+)](i)) and suppresses endothelial [Ca(2+)](i). The anion transport blocker probenecid helps retain fura 2 in the pericyte cytoplasm. DVR endothelia were accessed for membrane potential measurement by perforated-patch whole cell recording by using the pericyte-stripping technique and by turning nondigested vessels inside out with concentric micropipettes. By either method of access, 10 nM ANG II depolarized (n = 20) and 100 nM bradykinin hyperpolarized (n = 25) the endothelia. We conclude that isolated endothelia and pericytes remain functional for study of [Ca(2+)](i) responses and that ANG II and bradykinin receptors exist separately on each cell type.

We tested the hypothesis that constriction of descending vasa recta (DVR) is mediated by voltage-gated calcium entry. K(+) channel blockade with BaCl(2) (1 mM) or TEACl (30 mM) depolarized DVR smooth muscle/pericytes and constricted in vitro-perfused vessels. Pericyte depolarization by 100 mM extracellular KCl constricted DVR and increased pericyte intracellular Ca(2+) ([Ca(2+)](i)). The K(ATP) channel opener pinacidil (10(-7)-10(-4) M) hyperpolarized resting pericytes, repolarized pericytes previously depolarized by ANG II (10(-8) M), and vasodilated DVR. The DVR vasodilator bradykinin (10(-7) M) also reversed ANG II depolarization. The L-type Ca(2+) channel blocker diltiazem vasodilated ANG II (10(-8) M)- or KCl (100 mM)-preconstricted DVR, and the L-type agonist BayK 8644 constricted DVR. The plateau phase of the pericyte [Ca(2+)](i) response to ANG II was inhibited by diltiazem. These data support the conclusion that DVR vasoreactivity is controlled through variation of membrane potential and voltage-gated Ca(2+) entry into the pericyte cytoplasm.

We investigated the dependence of ANG II (10(-8) M)-induced constriction of outer medullary descending vasa recta (OMDVR) on membrane potential (Psim) and chloride ion. ANG II depolarized OMDVR, as measured by fully loading them with the voltage-sensitive dye bis[1,3-dibutylbarbituric acid-(5)] trimethineoxonol [DiBAC(4)(3)] or selectively loading their pericytes. ANG II was also observed to depolarize pericytes from a resting value of -55.6 +/- 2.6 to -26.2 +/- 5.4 mV when measured with gramicidin D-perforated patches. When measured with DiBAC(4)(3) in unstimulated vessels, neither changing extracellular Cl(-) concentration ([Cl(-)]) nor exposure to the chloride channel blocker indanyloxyacetic acid 94 (IAA-94; 30 microM) affected Psim. In contrast, IAA-94 repolarized OMDVR pretreated with ANG II. Neither IAA-94 (30 microM) nor niflumic acid (30 microM, 1 mM) affected the vasoactivity of unstimulated OMDVR, whereas both dilated ANG II-preconstricted vessels. Reduction of extracellular [Cl(-)] from 150 to 30 meq/l enhanced ANG II-induced constriction. Finally, we identified a Cl(-) channel in OMDVR pericytes that is activated by ANG II or by excision into extracellular buffer. We conclude that constriction of OMDVR by ANG II involves pericyte depolarization due, in part, to increased activity of chloride channels.

ANG II constricts descending vasa recta (DVR) through Ca(2+) signaling in pericytes. We examined the role of PKC DVR pericytes isolated from the rat renal outer medulla. The PKC blocker staurosporine (10 microM) eliminated ANG II (10 nM)-induced vasoconstriction, inhibited pericyte cytoplasmic Ca(2+) concentration ([Ca(2+)](cyt)) elevation, and blocked Mn(2+) influx into the cytoplasm. Activation of PKC by either 1,2-dioctanoyl-sn-glycerol (10 microM) or phorbol 12,13-dibutyrate (PDBu; 1 microM) induced both vasoconstriction and pericyte [Ca(2+)](cyt) elevation. Diltiazem (10 microM) blocked the ability of PDBu to increase pericyte [Ca(2+)](cyt) and enhance Mn(2+) influx. Both ANG II- and PDBu-induced PKC stimulated DVR generation of reactive oxygen species (ROS), measured by oxidation of dihydroethidium (DHE). The effect of ANG II was only significant when ANG II AT(2) receptors were blocked with PD-123319 (10 nM). PDBu augmentation of DHE oxidation was blocked by either TEMPOL (1 mM) or diphenylene iodonium (10 microM). We conclude that ANG II and PKC activation increases DVR pericyte [Ca(2+)](cyt), divalent ion conductance into the cytoplasm, and ROS generation.

We tested whether chronic ANG II infusion into rats affects descending vasa recta (DVR) contractility, synthesis of superoxide, or synthesis of nitric oxide (NO). Rats were infused with ANG II at 250 ng.kg(-1).min(-1) for 11-13 days. DVR were loaded with dihydroethidium (DHE) to measure superoxide and 3-amino-4-aminomethyl-2',7'-difluorofluorescein (DAFFM) to measure NO. Acute constriction of DVR by ANG II (0.1, 1, and 10 nM) was diminished, and NO generation rate was raised by chronic ANG II infusion. DHE oxidation by DVR from ANG II-infused rats was similar to controls and was significantly higher when NO synthesis was prevented with N(omega)-nitro-L-arginine methyl ester (L-NAME). The superoxide dismutase mimetic Tempol (1 mM) increased NO generation compared with controls. The increased synthesis of NO by chronic ANG II-treated vessels persisted in the presence of Tempol. DVR endothelial cytoplasmic Ca(2+) response to ACh was diminished by chronic ANG II treatment, but the capacity of ACh to increase NO generation was unaltered. We conclude that DVR generation of superoxide is not affected by chronic ANG II exposure but that basal NO synthesis is increased. DVR superoxide is unlikely to be an important mediator of chronic ANG II slow pressor hypertension in rats.

kidney; vasoconstriction; vasodilation; medulla; blood flow BLOOD FLOW TO THE MEDULLA of the kidney is maintained through the tonic and stimulated release of nitric oxide (NO). Intrarenal infusion of vasoconstrictors such as norepinephrine and angiotensin II (ANG II) cause vasoconstriction and a reduction of renal blood flow. In response to vasoconstrictor infusions, compensatory release of NO helps to preserve the small fraction of the total renal blood flow that reaches the medulla of the kidney (38,43,44). An important physiological role for the production of NO in the control of medullary blood flow has also been proposed based on the observation that NO synthase (NOS) inhibition leads to reduction of medullary blood flow, sodium retention, and hypertension (16,17,23). Calcium sensitive isoforms of NOS are expressed in a variety of structures within the renal medulla including the medullary thick ascending limb (mTAL), outer medullary collecting duct (OMCD), and descending vasa recta (DVR) endothelia (17,23). ANG II causes a compensatory increase in medullary NO generation (44) but reduces DVR endothelial intracellular calcium concentration ([Ca 2ϩ ] i ) and inhibits increases in [Ca 2ϩ ] i by bradykinin (BK) (25,31). Previous studies have shown that ANG II type 1 (AT 1 ) and 2 (AT 2 ) receptors are expressed in outer medullary vascular bundles (19,39). Motivated by this, we sought to determine which receptor subtype mediates the unusual suppressive effect of ANG II on DVR endothelial calcium signaling. We found that the AT 1 receptor blocker losartan inhibits the ANG II effect on DVR endothelia, but a high concentration of losartan is required. Interestingly, the ability of ANG II to reduce endothelial [Ca 2ϩ ] i elevation by BK and acetylcholine (ACh) is markedly enhanced when AT 2 receptors are blocked with PD-123319, implying an important role for AT 2 receptor activation to facilitate endothelial [Ca 2ϩ ] i signaling by vasodilators. We also examined the [Ca 2ϩ ] i transients induced by ANG II in the cytoplasm of isolated DVR smooth muscle/pericytes. In contrast to effects on endothelia, ANG II-induced pericyte [Ca 2ϩ ] i transients exhibited classical peak and plateau responses that were blocked by losartan but not PD-123319. METHODSIn vitro isolation of DVR. Studies were performed in 267 vessels from Sprague-Dawley rats (80-150 g body wt, Harlan) anesthetized before nephrectomy by an intraperitoneal bolus of thiopental (50 mg/kg body wt). DVR were microdissected as previously described from outer medullary vascular bundles and mounted on glass pipettes using a standard apparatus (ITM; San Antonio, TX) (26). The following buffer

Using fura 2-loaded vessels, we tested whether ouabain modulates endothelial cytoplasmic calcium concentration ([Ca(2+)](CYT)) in rat descending vasa recta (DVR). Over a broad range between 10(-10) and 10(-4) M, ouabain elicited biphasic peak and plateau [Ca(2+)](CYT) elevations. Blockade of voltage-gated Ca(2+) entry with nifedipine did not affect the response to ouabain mitigating against a role for myo-endothelial gap junctions. Reduction of extracellular Na(+) concentration ([Na(+)](o)) or Na(+)/Ca(2+) exchanger (NCX) inhibition with SEA-0400 (10(-6) M) elevated [Ca(2+)](CYT), supporting a role for NCX in the setting of basal [Ca(2+)](CYT). SEA-0400 abolished the [Ca(2+)](CYT) response to ouabain implicating NCX as a mediator. The transient peak phase of [Ca(2+)](CYT) elevation that followed either ouabain or reduction of [Na(+)](o) was abolished by 2-aminoethoxydiphenyl borate (5 x 10(-5) M). Cation channel blockade with La(3+) (10 muM) or SKF-96365 (10 muM) also attenuated the ouabain-induced [Ca(2+)](CYT) response. Ouabain pretreatment increased the [Ca(2+)](CYT) elevation elicited by bradykinin (10(-7) M). We conclude that inhibition of ouabain-sensitive Na(+)-K(+)-ATPase enhances DVR endothelial Ca(2+) store loading and modulates [Ca(2+)](CYT) signaling through mechanisms that involve NCX, Ca(2+) release, and cation channel activation.