INTRODUCTION

TOP ABSTRACT INTRODUCTION NO PRODUCTION AND THE... INITIAL EVIDENCE THAT NO... LOCALIZATION OF NO PRODUCTION... EVIDENCE FOR PARACRINE AND... IMPORTANCE OF MEDULLARY NO... INFLUENCE OF MEDULLARY NO... ACUTE COUNTERREGULATORY ACTION... RENAL MEDULLARY NO PROTECTS... GENETIC RAT MODEL OF... EVIDENCE THAT L-ARG UPTAKE... REFERENCES

THE RENAL MEDULLARY CIRCULATION is now recognized to be of importance for reasons that extend far beyond providing the economy of countercurrent exchange to concentrate large volumes of filtrate to produce small volumes of concentrated urine. It is now recognized that medullary blood flow (MBF) can play an important role in determining long-term levels of arterial pressure and that 15-30% reductions of blood flow to the renal medulla in the face of imperceptibly small changes of total renal blood flow can lead to the development of hypertension (16, 21). Evidence is reviewed showing that nitric oxide (NO) plays a unique role in the acute and chronic regulation of renal MBF, sodium homeostasis, and arterial pressure. More specifically, the role of NO in the regulation of MBF, in linking tubular metabolic needs with delivery of nutrients and as a counterregulatory system to protect the medulla from the consequences of underperfusion, are the focus of this review. The influence of NO on the renal cortex and such issues that relate to pressure-natriuresis, autoregulation of total renal blood flow, tubuloglomerular feedback, and glomerular filtration rate (GFR) regulation are important subjects of renal function that either have been reviewed by others (2, 29, 43, 55, 96) or are beyond the scope of this relatively brief review.

	NO PRODUCTION AND THE OVERALL REGULATION OF RENAL FUNCTION

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NO within the kidney is now known to contribute importantly to low renal vascular resistance (75, 115) and to be able to stimulate natriuresis and diuresis independent of renal perfusion pressure as reviewed by others (2, 5, 43, 50, 75, 104, 137). The slope of the pressure-natriuresis relationship has been shown to be reduced with the inhibition of renal NO synthase (NOS) activity (23, 28, 98). As reviewed recently by Liang and Knox (50) and by Ortiz and Garvin (80), there is evidence that NO reduces sodium reabsorption in all tubular segments, including the proximal tubule (50, 80), thick ascending limb (78, 79), distal tubules, and cortical collecting ducts (80), although medullary nephron segments and collecting ducts remain to be studied in detail. Studies have also demonstrated that tubuloglomerular feedback control of afferent arteriolar resistance is influenced by macula densa NO production, thereby enhancing autoregulation of renal blood flow, GFR, and renin secretion (30, 37, 53, 75, 122, 128).

	INITIAL EVIDENCE THAT NO PARTICIPATES IN THE ACUTE REGULATION OF MEDULLARY FUNCTION

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Evidence that an endothelial-derived relaxing factor (EDRF) within the renal medulla could play a role in medullary oxygenation was first reported by Biondi et al. (6). Functional evidence that basal levels of NO contribute to MBF resulted from a series of studies in our laboratory that examined the effects of a variety of vasoactive compounds on MBF. These compounds were assessed by direct delivery into the medullary interstitial space of rats by use of a small implanted catheter (51, 52), whereas MBF responses were determined using external and implanted optical fibers and laser-Doppler flowmetry techniques (52). Substances infused into the medullary interstitium became trapped and concentrated by the countercurrent vasa recta circulation, enabling responses to be determined independent of the cortex.

It was found that the most dramatic reduction of MBF was obtained with medullary infusion of the nonspecific NOS inhibitor N^G-nitro-L-arginine methyl ester (L-NAME) (62), a compound that reduced flow to this region by nearly 40% (61). No changes of cortical flow were observed. Similarly, infusions of bradykinin (57) and acetylcholine (62), NO-dependent vasodilators, also produced substantial increases of MBF. Several important conclusions were drawn from these in vivo studies in anesthetized rats. First, MBF could be modulated independently of cortical blood flow (CBF). Second, the medullary vasculature could respond to many of the same vasoactive compounds as arterioles in other regions of the body. Third, NO exerted a tonic influence on the renal medullary vasculature.

	LOCALIZATION OF NO PRODUCTION WITHIN VASCULAR AND TUBULAR STRUCTURES OF THE KIDNEY

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The extent to which the antinatriuresis associated with medullary NOS inhibition was a direct consequence of reduction of MBF (16, 102) or was related to changes of intrarenal paracrine/autocrine systems (95, 100) or was a direct consequence of tubular epithelial transport actions of NO (80, 136) awaited a better understanding of the intrarenal sites of NO production. Initial efforts to determine regional NO biosynthesis by determining tissue nitrite/nitrate and cGMP levels in slices of dissected renal zones, isolated glomeruli, and various cell cultures systems. These studies indicated that tissues of both the renal cortex and medulla were indeed capable of generating NO in abundant amounts (6, 10, 13, 25, 36, 69, 93, 108).

Recently, techniques have enabled discrete measurements of segmental tubular and microvascular measurements of NOS enzyme activity, expression of the mRNA and protein levels, and interstitial fluid NO concentrations ([NO]). NOS activity assays incubated tissue protein with L-[³H]arginine and appropriate cofactors and the conversion of L-arginine (L-Arg) to L-citrulline (Cit) was quantified by reverse-phase HPLC and radiochemical detection (63, 68). Evidence was obtained showing that the renal medulla is the major source of NO production in the rat kidney. Regional comparisons of NOS activity and [NO] are summarized in Fig. 1. NOS activity was found to be 26 times higher in the inner medulla and 4 times higher in the outer medulla than in the renal cortex of Sprague-Dawley rats (130). In vivo microdialysis techniques using oxyhemoglobin in the dialysate to trap tissue NO found that concentrations of interstitial fluid NO averaged nearly 105 ± 18 nmol/l in the renal medulla compared with 62 ± 16 nmol/l in the cortex (138). These observations conform with data showing that NOS I and NOS II mRNA levels were more than five times greater in the outer medulla of Brown Norway (BN) rats compared with cortex (132). Moreover, the protein expression levels of all three isoforms determined by Western blot analysis were observed to be three to five times higher in this region in BN rats (Cowley, unpublished observations), as also reported for NOS III in kidneys of Sprague-Dawley rats (77).

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Top: results are summarized for regional nitric oxide synthase (NOS) activity determined by HPLC as the formation of L-citrulline in homogenates of renal cortex (CX), outer medulla (OM), and inner medulla (IM). L-Arginine content in the reaction mixture of each tissue sample was adjusted to ~23 mmol/l by addition of exogenous cold L-arginine to OM and IM samples. * Significantly different (P < 0.05) from CX. Data regraphed from the original manuscript by Wu and colleagues (130). Bottom: regional concentration of NO, as determined by an oxyhemoglobin trapping technique, is shown. Data regraphed from the original manuscript by Zou et al. (136).

The capacity to produce NO is best represented by NOS activity, and studies have been carried to determine activity levels in microdissected glomeruli, tubular, and microvascular segments of the cortex and medulla of Sprague-Dawley rat kidneys (130). Inner medullary collecting ducts (IMCD) were found to contain the greatest levels of NOS activity (11.5 fmol Cit · mm¹ · h¹), greatly exceeding the activity found in the glomeruli (1.9 fmol Cit · glomerulus¹ · h¹). All other tubular elements exhibited significantly less NOS activity, including proximal convoluted tubule, pars convoluta, pars recta, cortical thick ascending limb, cortical collecting duct, outer medullary collecting duct, outer medullary thick ascending limb, and inner medullary thin limb. The medullary vasa recta bundles of the outer medulla contained relatively high levels of total NOS enzymatic activity (3.2 fmol Cit · mm¹ · h¹), exceeded only by that in the IMCDs. Only about one-third as much was found in the afferent arterioles and considerably less in the arcuate and interlobular arteries (63). Similar results were found when relative enzyme activity levels were expressed based on segment length or as total protein.

NOS I and NOS III mRNA was found to be expressed in both the glomeruli and in the vasa recta, whereas the IMCD contained mRNA for all three of the NOS isoforms (130). As initially observed by Terada et al. (121) and later confirmed by Ugie et al. (125) and ourselves (63), there appears to be little or no NOS mRNA for any of the known isoforms expressed in rat early proximal tubule, although NOS II mRNA was reported to be expressed in the S3 segment of the proximal tubule, based on in situ hybridization studies (121). Taken together, the data indicate that the NOS activity in IMCDs contributes importantly to the higher levels of interstitial [NO] observed in the medulla compared with the cortex.

	EVIDENCE FOR PARACRINE AND AUTOCRINE NO FUNCTION IN THE RENAL MEDULLA ("TUBULOVASCULAR CROSS-TALK")

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The use of fluorescent dyes for detection of NO and Ca²⁺ ions has begun to provide insights regarding the ability of medullary tubular epithelial cells to act as a paracrine NO source for the regulation of vasa recta function. The focus of these studies has been on the larger diameter vasa recta vessels of the outer medulla for reasons related to the unique structure and function of the medullary vasa recta circulation. Before the results of these studies are reviewed, it is important to first appreciate the microvascular anatomy of the renal medulla, as described in detail by others (3, 4, 40, 44, 64, 81-83, 127). In brief, it is recognized that blood flow to the medulla of mammalian kidneys is supplied by postglomerular microvessels derived from juxtamedullary glomeruli, the deep glomeruli of the inner cortex that receive their blood supply from the interlobular branches of the arcuate arteries, as described by Kaissling and Kriz (39). A photomicrograph of one of these juxtamedullary glomeruli (Glm) is shown in Fig. 2A. As seen in this figure, the efferent arterioles of these juxtamedullary glomeruli immediately radiate into the vasa recta capillary bundles of the outer medulla (OMDVR) that then coalesce into long descending vasa recta (DVR) loops supplying the inner medulla and papilla. Although the blood flow to the medulla emanates from the renal cortex (CX) and changes of cortical flow can importantly influence blood flow to the medullary region, there is evidence as presented in this review that flow to the medulla can be independently modulated. Regional regulation of MBF is important to supply nutrients and remove tubular reabsorbate. It is important to preserve the interstitial gradients for NaCl and urea established by the loops of Henle and collecting ducts for urine concentration.

View larger version (79K): [in this window] [in a new window]   Fig. 2.   Fluorescent microscopic image of outer medullary region with thick ascending limbs illuminated by superoxide (O)-sensitive fluorescent dye dihydroethidium (DHE) and with the vasa recta filled with latex microspheres (0.20 µm sized) as described in Ref. 21. The left kidney was removed, sliced into ~500-µm-thick sections, and imaged using a cooled charge-coupled device camera equipped with Nikon E600 fluorescence microscope to capture the fluorescent signals. Fluorescence was stimulated with 490-nm excitation filter and collected through 605/40-nm band path emission filter with images captured by MetaMorph software and enhanced by Adobe Photoshop software. A: a ×40 magnification of a juxtamedullary region of CX and OM region. Medullary thick ascending limbs (mTAL) are illuminated by DHE, and the outer medullary descending vasa recta (OMDVR) bundles are seen as a dark stripes filled with latex microspheres. B: ×100 magnification of vascular bundles (OMDVR) with surrounding mTAL. C: a light microscopic image of the pericytes of OMDVR (filled with microspheres) as seen in a micro-tissue strip isolated from the inner stripe of OM. [From Dickhout et al. (21).]

Medullary DVR vessels are unique in that they have characteristics of true capillaries with continuous endothelium but are also surrounded by smooth muscle remnants known as pericytes, as shown in Fig. 2C. The DVR pericytes contain -smooth muscle actin (88), and the ability of the pericytes to constrict and reduce the diameter of DVR has been demonstrated by Pallone and colleagues (86) using isolated perfused vasa recta vessels from the outer medulla. For example, these vessels have been shown to respond to vasoconstrictors such as ANG II, as shown in Fig. 3A (85), AVP (124), and endothelin (112), as well as vasodilators such as bradykinin (83), prostaglandin E₂ (112), and adenosine (111). The ability of the DVR to behave like small arterioles accounts for the ability of the medullary flow to be controlled independently from the cortical circulation when vasoactive substances are endogenously released or infused. This also accounts for the in vivo medullary flow responses observed when vasoactive agents are delivered into the renal medullary interstitium, such as AVP (72), norepinephrine (NE) (135), and ANG II (138). ANG II-induced reduction of MBF was also shown to be significantly enhanced by pretreatment with the cyclooxygenase inhibitor meclofenamate (61).

View larger version (50K): [in this window] [in a new window]   Fig. 3.   A: light micrograph of an OMDVR under basal conditions (top) and after addition of 10 nmol/l ANG II (bottom). Arrows indicate foci of vasoconstriction. (Courtesy of Dr. T. L. Pallone.) B: top shows a pseudo-color image of OMDVR pericytes in a control state after loading with the NO-sensitive dye 4,5-diaminofluorescein deacetate (DAF-2DA) in microtissue strips. Bottom: same cells after ANG II (1 µmol/l) administration in the suffusate demonstrating the increased NO concentration ([NO]). C: time course of intracellular [NO] ([NO]i) response of pericytes of the vasa recta with endothelium disrupted by microbeads perfusion in OMVDR to ANG II (1 µmol/l) measured by changes in DAF-2DA fluorescence intensity. Included in these micro-tissue strips were vasa recta surrounded by mTAL. * Bonferroni-corrected statistically significant increase of the means from 50 to 150 s after stimulation. [From Dickhout et al. (21).]

On the basis of these anatomic structures and blood flow responses of the outer medulla, studies were carried out to determine whether NO produced by the tubular epithelium of the medullary thick ascending limbs of Henle (mTAL) might act both as an autocrine factor within tubular epithelia and as a paracrine factor on surrounding structures. Although nephron segments are largely excluded from the central core of the vasa recta bundles, at the periphery of the bundle these vessels give rise to the dense capillary plexuses that perfuse the outer medulla. As shown in Fig. 2, A and B, these peripheral vasa recta are surrounded by medullary mTAL, and it was logical to examine whether these tubules could communicate information to the DVR via the release of NO.

The movement of NO in vivo has received far less attention than the biochemical reactions that control its rate of synthesis and degradation. The importance of the volatility, solubility, and diffusability of NO in determining its biological effects, however, has been studied in some detail and reviewed by Lancaster (46). The results have shown that under all physiological conditions (except when there is a gas-liquid interface as in the lung), NO exists completely as a dissolved nonelectrolyte. NO is slightly more soluble in aqueous solution than O₂, and in virtually all its biological actions NO is not a gas unless concentrations approach 1-2 mmol/l, a highly unlikely situation in vivo. The solubility of NO, although similar to O₂, is nearly nine times greater in organic solvents, so that NO concentrates in hydrophobic phases in tissues such as membranes, lipid inclusions, and lipoprotein complexes. The lipid bilayer of membranes therefore poses no barrier to the movement of NO in tissue. On the basis of mathematical modeling of the random diffusion of NO within tissues, it has been estimated that NO would be able to act at large distances from a single cellular source and thus affect an enormous number of other cells surrounding this single source (47). It has been estimated that NO could diffuse in tissues from 0.2 to 0.25 mm in 5-10 s (46), well within the range generally considered to be the tissue half-life of NO.

Microdialysis of renal interstitial fluid using oxyhemoglobin trapping has demonstrated relatively high levels of NO as well as nitrite concentrations in medullary interstitial fluid as shown in Fig. 1 (135, 136, 138). These NO levels were increased by intravenous infusion of L-Arg and decreased by L-NAME (136). Tubular and vascular segments of both the cortex and medulla therefore appear to be constantly exposed to NO that could serve a paracrine function.

Availability of novel fluorescent probes has enabled direct observation, at high temporal and spatial resolution, of the responses of intracellular NO and Ca²⁺ within renal tubular and epithelium vasa recta imaged from thin renal medullary tissue strips. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) has been quantified using fura 2-AM and changes of NO imaged using the NO-sensitive dye 4,5-diaminofluorescein deacetate (DAF-2DA). These techniques were initially used to study mechanisms of AVP-stimulated NO production in the IMCD epithelial cells. It was shown that AVP produced a rapid increase of [Ca²⁺]_i followed by a significant increase of intracellular NO within these cells (67). The NO response was abolished both by an NO scavenger, carboxy 2-(4-carboxyphenyl)-4,4,5,5,tetramethylimidazoline-1-oxyl-3-oxide, and by preincubation with the Ca²⁺-ATPase inhibitor thapsigargin. These results clearly demonstrated that NO acts as a signaling molecule in the IMCD.

As illustrated in Fig. 3, studies have also shown that NO produced within the mTAL can serve both a paracrine and autocrine function to control vascular tone of the surrounding vasa recta. The paracrine function was demonstrated when techniques were developed to distinguish the fluorescent signals of the contractile pericytes from the stronger signals of the underlying vasa recta endothelial cells. To do this, the fluorescent signals of the underlying endothelial cells were abolished by perfusing the kidneys with 0.2-µm latex microspheres before the removal of the microtissue strip from the outer medulla for study under the fluorescent microscope. This enabled a 10-fold increase in fluorescent signal gain of the detection unit for the acquisition of the less intense signals of the vasa recta pericytes without overexposure of fluorescent signals from the endothelial cells. The NO response to ANG II of an outer medullary vasa recta pericyte surrounded by mTAL in the microtissue strip loaded with NO-sensitive dye DAF-2DA was then measured (Fig. 3C).

These techniques were used to determine if the constrictor actions of ANG II on the vasa recta pericytes were buffered by NO produced by either tubular epithelial cells surrounding the vasa recta and/or by NO produced by endothelial cells within the vasa recta themselves (21). Using thin microtissue strips, ANG II (1 µmol/l) produced an increase of intracellular [NO] ([NO]_i) in the vasa recta pericytes only when the vessels were adjacent to the mTAL. Pericytes of completely isolated vasa recta without surrounding mTAL showed a rapid peak increase in [Ca²⁺]_i (21), consistent with observations of Reinhardt et al. (97). Importantly, however, DVR pericytes did not show any increase of NO production either in response to ANG II or with administration of a Ca²⁺ ionophore, indicating a lack of Ca²⁺-sensitive NO production in vasa recta pericytes. Using isolated vasa recta with intact endothelium, ANG II was found to reduce Ca²⁺ within the endothelial cells as was also observed by Pallone and colleagues (85, 86) and failed to increase NO, although the Ca²⁺ ionophore was able to increase NO in these endothelial cells. Tubular epithelial cells responded quite differently in that increases of NO were observed in single isolated mTAL in response to both ANG II and Ca²⁺ ionophore. Although it is evident that the concentrations of ANG II used in these studies far exceeded levels in the systemic circulation, it appears that intrarenal levels may be as high as 10⁹ to 10¹⁰ mol/l (73, 107) so the medullary structures may be exposed to nanomolar levels of ANG II (75). Pallone et al. (85) observed isolated DVR diameter reductions at concentrations of 10 nmol/l (see Fig. 3A).

The autocrine function of NO produced in rat thick ascending limb (TAL) segments was established by Oritz and Garvin (78), who demonstrated in perfused isolated tubules that endogenously produced NO inhibited HCO₃ transport. Although it is not stated in this study whether TAL segments were obtained from the cortex or medulla, it is likely that these events take place in both segments.

On the basis of these studies, autocrine/paracrine concepts emerged as summarized in Fig. 4. ANG II increases [NO]_i in pericytes only when the vasa recta vessels are surrounded by renal tubular segments, mostly mTAL in this case. Neither the endothelium nor pericytes can directly increase NO production in response to ANG II. ANG II increases intracellular calcium concentration in the pericytes of the DVR, apparently as part of its constrictor action on these vessels. This response is buffered by NO diffusing from tubular elements surrounding vasa recta within the outer medulla, a paracrine function that we have called tubulovascular cross-talk. The close proximity of mTAL to the vasa recta bundles in the outer medulla (~50-150 µm) suggests that these tubular elements would be of greater importance in this paracrine cross-talk than the outer medullary collecting ducts, which are of a greater distance to the vessels (~150-250 µm) (39, 44, 49). The extent to which hemoglobin within the vasa recta capillary circulation would modify the in vivo NO diffusion profile is unclear. However, NO derived from mTAL is not exposed to hemoglobin before reaching the pericytes of the vasa recta. Because there is considerable interstitial space and fluid surrounding the individual vasa recta to provide NO to the pericytes (~10- to 30-µm distances separating the vasa recta; personal observation), intravascular hemoglobin would not be expected to play a major role in buffering the medullary interstitial NO gradients. The diameter of the entire vasa recta bundle does not exceed the estimated diffusion distance of NO within biological fluids (0.2-0.25 mm) as indicated above (46, 47). Finally, although the deep vasa recta of the inner medulla remain to be studied, it is likely that these vessels may be controlled in a similar fashion by the high levels of NO produced by the epithelial cells of the IMCD, because pericytes have been shown to exist surrounding these vasa recta, although less densely distributed (88).

View larger version (50K): [in this window] [in a new window]   Fig. 4.   Schematic summary of the observed actions of ANG II on NO levels and intracellular Ca2+ concentration ([Ca2+]i) in outer medullary vascular bundles. ANG II increases [Ca2+]i in pericytes of the descending vasa recta and reduces [Ca2+]i in endothelium of the descending vasa recta. ANG II also increases [NO]i in the pericytes of the descending vasa recta but only when these cells are in proximity to the mTAL surrounding the outer medullary vascular bundles. ANG II increases [Ca2+]i and [NO]i in mTALs even when these tubules were studied in isolation. These observations indicate that ANG II exerts a constrictor effect on the descending vasa recta by direct action on pericytes and that this constrictor action is buffered by NO diffusing from mTALs to the pericytes of the descending vasa recta. [Schematic modified from original in Dickhout et al. (21).]

	IMPORTANCE OF MEDULLARY NO PRODUCTION IN THE LONG-TERM CONTROL OF MBF AND ARTERIAL PRESSURE

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The first evidence that basal NO production played an important role in determining arterial blood pressure arose from observations that chronic oral or intravenous administration of NOS inhibitors such as L-NAME or N^G-monomethyl-L-arginine (L-NMMA) produced hypertension (28, 32, 55, 123). It was unclear, however, whether the observed hypertension was primarily a result of generalized systemic vasoconstriction, an effect of NO reduction in brain, or a primary consequence of reduced renal excretory function. Studies were carried out to determine whether medullary NO production per se played an important role in the long-term regulation of renal MBF, sodium excretion, and arterial pressure. L-NAME was infused chronically into the renal medullary interstitium of Sprague-Dawley rats for 5 days. As shown in Fig. 5, this renal interstitial (ri) infusion of L-NAME resulted in a sustained reduction of renal MBF (~30%) with no measurable changes of cortical flow as determined daily in unanesthetized rats with chronically implanted optical fibers. This reduction of MBF was associated with an immediate reduction of sodium excretion, retention of sodium, and the development of hypertension (59). This demonstrated that reduction of NO production in the medulla alone could reduce MBF and produce hypertension.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Changes in mean arterial pressure (MAP), renal medullary blood flow (MBF), and cortical blood flow (CBF) in conscious, uninephrectomized Sprague-Dawley rats during renal medullary interstitial (ri) infusion of NG-nitro-L-arginine methyl ester (L-NAME; 120 µg/h). Blood flow was measured by laser-Doppler flowmetry with implanted optical fibers. * Significant difference from 3rd control day. [Regraphed from original data (59)].

Consistent with these observations, it was also found that L-NAME administered in small doses by chronic intravenous infusion reduced MBF alone, independently of any changes in CBF, and this again led to sodium retention and hypertension (71). It had been assumed that hypertension resulting from systemically administered L-NAME was probably related to a reduction of total renal blood flow (75) and an overall increase in systemic vascular resistance related to vascular and central nervous system NOS inhibition. Together these studies with both systemic (intravenous) and renal medullary (ri) delivery of L-NAME demonstrated that the production of NO specifically within the medulla was of primary importance in the long-term regulation of MBF and sodium homeostasis and that reduction of NOS activity in the renal medulla alone could produce chronic hypertension.

	INFLUENCE OF MEDULLARY NO PRODUCTION ON BLOOD PRESSURE SALT SENSITIVITY

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Consistent with the influence of NO on tubular sodium reabsorption summarized above, it was found that systemic administration of nonspecific NOS inhibitors resulted in a sodium-dependent form of hypertension (28, 55, 70, 123). The extent to which reduction of medullary NO independent of the renal cortex could produce blood pressure salt sensitivity was determined by a series of studies In our laboratory. The effects of inhibiting two of the specific NOS isoforms, NOS I and NOS II, were examined. A salt-dependent form of hypertension occurred in Sprague-Dawley rats when an antisense oligonucleotide complementary to the code of the initiating region of the mRNA for NOS I synthesis was infused chronically into the medullary interstitial space (56). Similar responses were observed using a mechanistically different inhibitor of NOS I, 7-nitroindazole (7-NI) (56). The antisense probe reduced NOS I synthase activity by 53% and the 7-NI by 37%. In both cases, mean arterial pressure rose over 3 to 5 days to ~15 mmHg above resting control levels in rats receiving a high (4%)-salt diet, with no changes in pressure being observed in rats receiving the low-salt diet.

In another study, the effects of chronically inhibiting the inducible NOS II isoforms were examined (60). Chronic intravenous infusions of aminoguanidine in Sprague-Dawley rats that resulted in a 49% reduction of Ca²⁺-independent NOS (inducible NOS) in the renal medulla, produced a salt-dependent form of hypertension with mean arterial pressure rising nearly 15 mmHg when these rats were fed a high-salt (4%) diet. The results also suggested that these events occurred independently of renal vascular changes, because intravenous infusion of this inhibitor at the same dose in anesthetized rats resulted in no significant changes in mean arterial pressure, CBF, or MBF, whereas urine flow and sodium excretion were significantly decreased (60).

Thus it appears that reduction of NO production within the renal medulla alone can lead to a salt-sensitive form of hypertension. Tan et al. (120) observed hypertensive responses in the absence of any measurable changes in renal plasma flow or GFR with chronic intravenous administration of aminoguanidine in salt-resistant Dahl R rats exposed to a high-salt diet, consistent with predominant tubular effects. Recent work by Kakoki et al. (42) and Mattson et al. (60) using a variety of NOS isoform-specific inhibitors indicated that NO derived from NOS I and/or NOS II has minimal effect on blood flow in the renal medulla (42, 60). The results also suggest that the enhanced salt sensitivity with NOS inhibition may be largely a consequence of a reduction of NOS I and NOS II activity within the deep nephrons of the renal medulla. The role of NOS II, however, remains controversial given the lack of complete specificity of these inhibitors. The antinatriuretic effects of aminoguanidine have not been consistently observed by others (43), and firm conclusions regarding the specific roles of NOS II in the regulation of medullary function await final resolution.

The lack of availability of NOS III-specific inhibitors has precluded pharmacological approaches to examine this isoform. One would expect, however, that specific reductions of endothelial NOS would be accompanied by a lowering of MBF and chronic hypertension as seen with L-NAME administration. Huang et al. (33) observed hypertension in NOS III-knockout mice, demonstrating the importance of NOS III in long-term blood pressure regulation, but this study did not address the role of NOS III in the renal medulla. Inasmuch as NOS III is found in both endothelium of the vasa recta and the epithelial cells of mTAL and IMCD (see below), the NO produced by this NOS isoform could influence both tubular sodium reabsorption and MBF. Thus it will not be so easy to separate the relative contributions of NO from these specific regions upon the long-term control of arterial pressure.

The role of medullary NO in response to changes of sodium intake and the extent to which such changes participate in the regulation of MBF remain unclear. Studies in chronically instrumented Sprague-Dawley rats in our laboratory found no changes in MBF as daily sodium intake was increased from 0.4 to 4.0% (27). However, rats fed a high-sodium diet (4.0%) exhibited an increase in NOS-immunoreactive protein for all isoforms in the medullary tissue (58). Ni and Vaziri (76) observed a decrease in medullary protein expression of NOS I and II after 3 wk of an 8% sodium diet. Others found no change in medullary mRNA expression of any of the NOS isoforms (98, 113).

	ACUTE COUNTERREGULATORY ACTION OF RENAL MEDULLARY NO ON CIRCULATING VASOCONSTRICTORS

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The vascular counterregulatory functions of NO were first suspected, but the data supporting this role for NO were not initially forthcoming. Reduction of tissue NO production with NOS inhibition was found by some investigators to enhance ANG II-induced reduction of renal blood flow (1) and to potentiate the vasoconstrictor effects of ANG II in isolated perfused kidneys (105). Similar findings were obtained using the in vitro perfused juxtamedullary preparation (35) and in isolated perfused afferent arterioles (37). Others found no evidence of enhanced vasoconstriction to either ANG II or NE after NOS inhibition using a perfused split kidney juxtamedullary preparation when vessel diameter was restored to normal by sodium nitroprusside (35), or in studies using an in vitro hydronephrotic kidney preparation (87).

Considerable evidence has now emerged supporting the view that NO does serve an important counterregulatory role within the renal medullary circulation. In this regard, ANG II has been studied in some detail. There is abundant evidence that ANG II can stimulate NO production in many cell types, as shown by measurements of nitrate and cGMP production in neural, cardiac, vascular, and renal cells (8, 14, 34, 45, 106, 126, 133). ANG II also increases the renal excretion of nitrate and nitrite (20) and cGMP concentrations in renal cortical interstitial fluid as determined by microdialysis (110).

Results of these earlier studies were unable to provide conclusive evidence that NO served as an important counterregulatory system to vasoconstrictor agents in the renal medulla, because neither tissue [NO] nor MBF were measured directly. Both of these are needed to ascertain changes in local NO production. Blood flow measurement is important because [NO] alone need not reflect increased NO production, because medullary interstitial tissue and fluid [NO] can be hemodynamically influenced by the rate of blood flow through the countercurrent vasa recta circulation ("washout") and by shunt pathways in the outer medulla, as known to be the case with oxygen gradients.

Studies were therefore carried out to determine the in vivo effects of ANG II on both MBF and interstitial fluid [NO] (via microdialysis oxyhemoglobin trapping technique; Ref. 136). As shown in Fig. 6A, intravenous infusion of a subpressor dose of ANG II (5.0 ng · kg¹ · min¹) produced no reduction of CBF, MBF, or medullary PO₂ levels. This level of infused ANG II, however, did produce nearly a 150% rise of [NO] within the medullary interstitial fluid (138). This ANG II-induced rise of tissue fluid [NO] was abolished when medullary NOS activity was blunted in these same rats by medullary infusion of a low dose of L-NAME, a dose carefully determined to have no measurable effect on basal MBF. In the absence of the ANG II-stimulated increase of [NO], there was a reduction of both MBF and tissue PO₂ of nearly 30%. These results demonstrated the importance of the NO counterregulatory actions on the regulation of MBF and oxygen delivery. Since Navar et al. (74) found that, even under normal conditions, medullary tissue ANG II concentrations are significantly higher than those of the renal cortex, the regional generation of NO within the medulla could be of substantial importance even in reducing the basal vasoconstrictor actions of ANG II in this region of the kidney.

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Left: effect of intravenous (iv) infusion of L-NAME () or saline (l) on ANG II response of MAP, CBF, and MBF and medullary oxygenation (PmO2) after 2 control periods (C1 and C2). CBF and MBF were measured by using laser-Doppler flowmetry, and medullary tissue oxygen was measured by microelectrode polargraphic technique. * Significant difference from the control periods (P < 0.05). [Reprinted with permission from Zou et al. (138).] Right: using a similar study design, the effect of iv infusion of L-NAME () or saline () on norepinephrine (NE) response of MAP, CBF, MBF, and PmO2 is summarized. [From Zou and Cowley (135).]

The effects of NE on medullary NO release have also been examined. Several studies found that NE can induce NO release through the ₂-receptors (35, 68, 135). ₂-Activation appears to increase the influence of NO in the glomeruli and proximal tubules of rats (130), and it has been shown that the vasculature becomes more sensitive to ₂-receptor stimulation with chronic NOS inhibition (31). As summarized in Fig. 6B, studies have demonstrated the in vivo effects of NE on the medullary circulation. In response to 1-h intravenous infusions of subpressor amounts of NE (0.1 µg · kg¹ · min¹), medullary [NO] increased 43% (135). This response was inhibited with a selective ₂-adrenergic inhibitor (135). As was observed in the ANG II studies (138), when medullary NOS activity was blunted by medullary infusion of a low dose of L-NAME, the NE-induced rise of tissue [NO] was abolished and a reduction of both MBF and tissue PO₂ of nearly 30% was observed (135). These results again demonstrated the importance of the NO counterregulatory actions on the regulation of MBF and oxygen delivery.

Similar findings were also obtained in studies using subpressor amounts of AVP (2 ng · kg¹ · min¹) in which a 35% increase of medullary NO was observed (89), a response mediated via the V₂-type receptor. These observations were consistent with the direct observations that AVP stimulated NO production in the epithelial cells of inner medullary collecting ducts (IMCD) as determined by fluorescent microscopy (67) and other reports that AVP stimulated increases of urinary cGMP concentrations (103) and that the inhibition of NOS acutely enhanced systemic pressor responses to AVP (35, 109).

Together, these studies demonstrated two important aspects of medullary NO production. First, subpressor elevations of circulating ANG II, NE, and AVP result in a substantial increase of renal medullary NO production. Second, one of the important functions of medullary NO production is related to the counterregulatory actions of this NO in response to stimulation by vasoconstrictor stimuli. The rise of medullary NO stimulated by these vasoconstrictor compounds significantly buffers both the reduction of MBF and tissue PO₂.

	RENAL MEDULLARY NO PROTECTS AGAINST CHRONIC ANG II-, NE-, AND AVP-INDUCED HYPERTENSION

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On the basis of the acute observations described above, a series of studies was carried out in chronically instrumented rats to determine whether the stimulation of NO by vasoconstrictor compounds would buffer or prevent chronic reductions of MBF and hypertension. Studies determined whether selective blunting of NOS activity in the renal medulla would result in a hypersensitivity to small elevations of circulating vasoconstrictor compounds and consequently result in hypertension. A dose of L-NAME (75 µg · kg¹ · h¹) was used that did not reduce basal MBF >5% when administered into the medullary interstitial space chronically for 2 wk (118). The same dose of L-NAME that significantly attenuated acute increases of medullary [NO] produced by intravenous infusions of subpressor amounts of ANG II, NE, and AVP (116-118). Doses of ANG II (3.0 ng · kg¹ · min¹), NE (0.1 µg · kg¹ · min¹), and AVP (2.0 ng · kg¹ · min¹) were determined that when administered intravenously did not result in elevations of mean arterial pressure >5 mmHg when infused for 5-7 days to normal Sprague-Dawley rats (see Fig. 7). Each compound was then infused intravenously, whereas the nonhypertensive dose of L-NAME was infused chronically into the medullary interstitium to blunt medullary NOS activity.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Influence of NO blockade by L-NAME delivered chronically into the renal interstitium on the blood pressure response to vasoconstrictors given intravenously. MAP was normalized to allow comparison between the individual studies using nonpressor doses of ANG II (116), NE (118), and arginine vasopressin (117). , Saline ri; , L-NAME ri.

In the normal state (e.g., an absence of medullary L-NAME infusion), AVP infusion displayed a well-known response pattern with an initial rise of arterial pressure for the first several days of intravenous infusion and then returning to normal (19, 92). Chronic hypertension had never been observed previously with chronic elevations of plasma AVP in rats (19, 92), humans (48), or dogs (114). However, as seen in Fig. 7, in rats that received the subpressor chronic medullary infusion of L-NAME, AVP led to a sustained elevation of blood pressure (117). Similar studies were carried out to determine the responses to chronic intravenous infusions of subpressor doses of ANG II (116) and NE (118). Also shown in Fig. 7, hypertension occurred associated with sustained reductions of MBF (not shown) only when medullary NOS activity was blunted by the medullary interstitial infusion of L-NAME (116, 117).

The medullary circulation of rats therefore appears to be well protected from both the acute and chronic vasoconstrictor actions of small increases in circulating levels of ANG II, NE, and AVP. Although many of the details of these mechanisms remain to be elucidated, these studies show that NO-mediated responses play a key role in protecting the renal medulla from excessive vasoconstriction and chronic hypertension.

Other medullary systems may also be involved in protecting this region from underperfusion. On the basis of acute studies in anesthetized rats, one of the other medullary counterregulatory systems may be prostaglandin release. We have observed that rats pretreated with a cyclooxygenase inhibitor (meclofenemate) exhibited a significant reduction of medullary flow with subpressor intravenous infusion of ANG II (61). Similar responses have been observed with kinin receptor antagonists (24, 99). It has now also been found that the hemoxygenase enzymes are present at significantly greater concentrations in the renal medulla than in the cortex, and the production of medullary CO via the hemoxygenase pathway may participate in the control of flow to this region (134). It appears therefore that the renal medulla contains a number of redundant systems that could serve to protect blood flow to this region. However, only the NO system at this time has been sufficiently studied to conclude that it indeed represents a feedback control system for the long-term regulation of blood flow to the renal medulla.

The NO counterregulatory responses are of seemingly great importance in maintaining an adequate blood flow to the outer medulla. The delivery of oxygen to the mTAL is dependent on this blood flow. There is a high rate of O₂ utilization due to the high metabolic ion transport activity of the thick ascending limbs of Henle that absorbs nearly 25% of the filtered sodium load. Blood flow to the renal cortex per gram of tissue is substantially higher in the cortex (700 ml · min¹ · 100 g¹) compared with the inner medulla (100 ml · min¹ · 100 g¹) (9); the hematocrit of vasa recta blood is less than half (26%) of that of arterial blood (101). The countercurrent flow in the vasa recta and shunting of O₂ between descending and ascending vasa recta account in part for the falling gradient of PO₂ observed from cortex to the tip of the papilla (6, 10, 12). These features result in the observed PO₂ levels of nearly 45 mmHg in the outer medulla falling progressively to <10 mmHg in the inner medulla and tip of papilla (10, 11, 138). This contributes to the cells in the deepest zone of the outer medulla receiving less oxygen, making them more susceptible to ischemic injury than cells situated in the cortex (12). The vulnerability of the medulla to underperfusion is expressed by tubulointerstitial injuries in the juxtamedullary region seen early in the development of hypertension in rats (22, 26, 38, 94, 131) and humans (7).

	GENETIC RAT MODEL OF REDUCED MEDULLARY NO PRODUCTION: THE DAHL SS RAT

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It has been found that salt-sensitive Dahl rats (SS) exhibit a reduced capacity to generate NO within the renal medulla. The outer medulla NOS enzyme activity was found to be nearly one-third less in SS rats than in normotensive salt-resistant BN rats, although NOS activity was nearly the same in the inner medulla (119; Fig. 8B). Consistent with these findings, the mRNA and protein for each of the three NOS isoforms was also less in the outer medulla of SS rats compared with BN control rats (Fig. 8, A and C; 119, 132). Most importantly, although basal medullary [NO] was not significantly lower in SS compared with BN rats, the ability of ANG II to stimulate an increase of medullary NO was greatly blunted in SS rats as shown in Fig. 8D (119). These observations predict that the renal medullary circulation of the SS inbred rat would be hypersensitive to small elevations of circulating vasoconstrictor agents due to blunting of the NO counterregulatory system.

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Summary of studies demonstrating the reduced capacity of the Dahl salt-sensitive (SS) rat to generate NO within the outer medulla compared with the Brown Norway (BN). A: significantly reduced mRNA expression (determined by Northern blot) of the 2 detectable NOS isoforms in SS rats compared with the BN rats (132). NOS activity in the outer medulla of SS rats is also significantly decreased as depicted in B (119). C: results of the Western blot analysis of protein expression of the 3 NOS isoforms in which all 3 isoforms were significantly lower in the SS rat compared with BN rat (119). D: significant increase in the concentration of NO in the outer medulla after a 1-h intravenous infusion of ANG II in the BN rat, with the SS rat showing no change from control (119). Control [NO] for SS rat was 89 ± 3 and 87 ± 3 nmol/l for the BN rat. * Significant difference between the SS and BN in A-C. * Significant difference from the pre-ANG II control in D.

Studies were carried out to determine if SS rats resembled Sprague-Dawley rats, in which medullary NOS activity was blunted by selective delivery of L-NAME chronically into the medullary interstitium. As summarized in Fig. 9A, chronic intravenous infusion of ANG II (3.0 ng · kg¹ · min¹) failed to induce a rise of mean arterial pressure in the salt-insensitive BN rats as had been found earlier in Sprague-Dawley rats (119). However, this same dose of systemically delivered ANG II resulted in sustained hypertension in SS rats. It is important to recognize that both the SS and BN rats were maintained on a low (0.4%) salt intake throughout the study. These results were consistent with the acute responses of MBF to low-dose acute intravenous ANG II infusions (5.0 ng · kg¹ · min¹). That is, ANG II significantly reduced MBF in SS rats, but not in BN rats (119). The ability of the BN rats to buffer ANG II-induced reductions of MBF but not in SS rats was also consistent with observations that ANG II induced a significant increase of medullary interstitial [NO] of BN rats, but failed to do so in SS rats (119).

View larger version (15K): [in this window] [in a new window]   Fig. 9.   Change in MAP in SS and BN rats is shown after chronic intravenous infusions of a nonpressor dose of ANG II (119) or AVP (132). Control pressures were normalized (presented as %) to better contrast the results from the 4 groups studied.

Similar studies were carried out with chronic intravenous infusion of AVP (132). As shown in Fig. 9B, mean arterial pressure in SS rats maintained on a low (0.4%)-salt diet increased from a control of 127 mmHg to an average of 147 mmHg after 14 days of AVP infusion (2.0 ng · kg¹ · min¹). These were remarkable observations, because it was the first time that AVP produced sustained hypertension without pharmacological manipulations or surgical reductions of renal mass (54). Northern blot analysis of renal outer medullary tissue in this study demonstrated that SS rats maintained on a low (0.4%)-salt diet expressed NOS I and NOS II mRNA levels that were significantly less than that found in BN rats maintained on the same low salt intake (132; Fig. 8A).

Taken together, the results of studies in both Sprague-Dawley rats receiving a medullary infusion of a low dose of L-NAME and in SS rats indicate that an impaired NO counterregulatory system in the renal medulla results in reduction of blood flow to this region and an increased susceptibility to develop hypertension in face of small elevations of circulating vasoconstrictor compounds such as ANG II, AVP, and NE.

	EVIDENCE THAT L-ARG UPTAKE IN THE RENAL MEDULLA PARTICIPATES IN THE REGULATION OF NO PRODUCTION

TOP ABSTRACT INTRODUCTION NO PRODUCTION AND THE... INITIAL EVIDENCE THAT NO... LOCALIZATION OF NO PRODUCTION... EVIDENCE FOR PARACRINE AND... IMPORTANCE OF MEDULLARY NO... INFLUENCE OF MEDULLARY NO... ACUTE COUNTERREGULATORY ACTION... RENAL MEDULLARY NO PROTECTS... GENETIC RAT MODEL OF... EVIDENCE THAT L-ARG UPTAKE... REFERENCES

Both oral and intravenous administration of the NOS substrate L-Arg in large amounts have been shown to be capable of preventing salt-induced hypertension in SS rats (15, 32, 90, 91). It has also been found in anesthetized rats that pressure-natriuresis and transmission of perfusion pressure into the renal interstitium were normalized by long-term L-Arg treatment (90, 91). These observations led us to hypothesize that the major antihypertensive actions of L-Arg in SS rats could be mediated largely through actions of this NO precursor, specifically within the renal medulla. A threshold dose of L-Arg was found that when infused into the medullary interstitium increased renal MBF without altering CBF in anesthetized SS rats. This dose (300 µg · kg¹ · min¹) was then infused chronically into the renal medullary interstitium of SS rats to determine the effects of this regional L-Arg administration on salt-induced hypertension in these rats (65, 66). Remarkably, as daily salt diet was increased from 0.4% to 4.0%, mean arterial pressure remained nearly unchanged over 1 wk in SS rats receiving the medullary infusion of L-Arg (Fig. 10). In contrast, in SS rats receiving the saline vehicle control infusion, mean arterial pressure increased from a control level of 120 to 165 mmHg over the same time period. The same dose of medullary infused L-Arg that prevented salt-induced hypertension in SS rats failed to blunt the development of hypertension when administered intravenously to SS rats. It was found that SS rats receiving the high-salt diet exhibited a significant reduction of MBF (65). This reduction of flow to the renal medulla of the SS rat was prevented by chronic medullary interstitial delivery of L-Arg with no rise of arterial pressure observed. These data, as summarized in Fig. 10, indicated that the prevention of salt-sensitivity in SS rats was due specifically to the action of L-Arg on the renal medullary function and that SS rats may have a deficit of medullary substrate availability that could account in part for the observed reduction in NO production in this region of the kidney.

View larger version (21K): [in this window] [in a new window]   Fig. 10.   effects of renal medullary interstitial infusion of L-Arg (300 µg/kg/min) in SS rats on high-salt diet induced changes in MAP, CBF, and MBF. SS rats infused with L-Arg ri are indicated by . SS rats not infused with L-Arg but undergoing the same change in diet are indicated by . [These data are regraphed from the original manuscript by Miyata and Cowley (65).]

Studies carried out to characterize the pathway of cellular uptake of L-Arg determined the influence of cellular L-Arg uptake on NOS enzymatic activity in IMCD cells from Sprague-Dawley rat kidneys (129). It was found that L-Arg uptake was predominantly mediated by a high-affinity y+ transporter system, a sodium-independent, trans-stimulated cationic transporter encoded by the CAT 1 gene. The y+ transporter system consists of a family of four genes, designated as CAT1, CAT2A, CAT2B, and CAT3, but the only transporter found in IMCD was CAT1. Biochemical transport studies using L-[³H]arginine found that the L-Arg transport system was saturable and inhibited by the cationic amino acids L-lysine, L-homoarginine, and L-ornithine, but was unaffected by the neutral amino acids L-leucine, L-serine, and L-glutamine. Both L-ornithine and L-lysine inhibited NOS enzymatic activity in a dose-dependent manner in IMCD cells, supporting the important role of L-Arg transport for NO production by this tubular segment in the renal medulla.

To determine whether chronic inhibition of L-Arg uptake in the renal medulla would reduce [NO] and produce hypertension, the effects of renal medullary infusion of cationic amino acids and an antisense oligonucleotide for CAT1 were determined in Sprague-Dawley rats (41). These studies demonstrated that chronic inhibition of L-Arg uptake in the renal medulla with a CAT1 antisense oligonucleotide or by coadministration of other competing cationic amino acids led to a sustained reduction of NO and the development of hypertension (41). In contrast, chronic intravenous infusion of the same dose of these cationic amino acids did not change NO in the medulla nor alter arterial pressure. These studies provide evidence that the in vivo production of NO and the regulation of MBF of the rat kidney are partially dependent on the cellular uptake of L-Arg by a y+ transport system. It remains to be determined if this system is altered in the Dahl salt-sensitive model of hypertension.

Perspectives