Vasa Recta Research Articles

Cycloxygenase-2 is expressed in vasculature of normal and ischemic adult human kidney and is colocalized with vascular prostaglandin E2 EP4 receptors.

The study was performed to elucidate the distribution and cellular localization of cyclooxygenase (COX)-2 in human kidney and to address localization of downstream targets for COX-derived prostanoids. Cortex and outer and inner medulla tissue were obtained from control kidneys (cancer specimens), kidneys with arterial stenosis, and kidneys of patients who received angiotensin II inhibition or acetylsalicylic acid. Ribonuclease protection assay and Western blot test revealed that COX-1 and -2 mRNA and protein were expressed in all regions of human kidney (mRNA ratio, cortex:outer medulla:inner medulla COX-1 1:3:20 and COX-2 1:1:3). In adult kidney, immunohistochemical labeling for COX-2 was associated with smooth muscle cells in pre- and postglomerular vessels and with endothelium, particularly in vasa recta and medullary capillaries. Western blot test confirmed COX-2 expression in renal artery. COX-2 had a similar localization in fetal kidney and was additionally observed in Henle's loop and macula densa. Human tissue arrays displayed COX-2 labeling of vascular smooth muscle in multiple extrarenal tissues. Vascular COX-2 expression was significantly increased in kidneys with arterial stenosis. COX-1 was colocalized with microsomal prostaglandin E(2) synthase (PGES) in collecting ducts, and PGES was also detected in macula densa cells. Vascular COX-2 was colocalized with prostaglandin E(2) EP4 receptors but not with EP2 receptors. Thus, renovascular COX-2 expression was a constitutive feature encountered in human kidneys at all ages, whereas COX-2 was seen in macula densa only in fetal kidney. Vascular COX-2 activity in human kidney and extrarenal tissues may support blood flow and affect vascular wall-blood interaction.

Functional and Structural Postglomerular Alterations in the Kidney of Prehypertensive Spontaneously Hypertensive Rats

The kidney plays a major role in the development of hypertension. Following the Borst‐Guyton theory of an altered set‐point for fluid and electrolyte homeostasis we aim to investigate functional and structural renal parameter during the development of hypertension. Therefore we focus on counter current exchange related factors. We compared 4 and 8 weeks old Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) concerning basic renal parameters as creatinine and phosphorus clearance and urinary osmolality. Mean arterial pressure (MAP) was measured intra‐arterially. Vasa recta were investigated using immunohistochemistry for α‐smooth‐muscle actin (ASMA) and plastification for geometric analyses. Blood pressure was not yet significantly elevated in SHR at 4 weeks but at 8 weeks it was higher in SHR (116 ± 7 vs. 102 ± 4 mm Hg; p < 0.01). Kidney weight/body weight ratio was lower in SHR at both ages. In 4 weeks old SHR, phosphorus clearance and urinary osmolality were decreased compared to WKY [0.02 ± 0.01 vs. 0.05 ± 0.02 (ml/min*100 g BW) p < 0.03; 14.2 ± 2.2 vs. 18.9 ± 2.9 (osmol/kg*24 h urine) p < 0.05] indicating reduced tubular reabsorption. At 8 weeks phosphorus clearance and urinary osmolality were comparable to WKY. α‐Actin was found in vasa recta in a 4‐times higher degree in SHR with a predominant location in the outer medulla. Radii of vasa recta in the outer medulla decreased during development. In plastificated sections vasa recta of SHR revealed sphincter‐like pattern. Functional and structural alterations related to the counter current exchanger are already evident in prehypertensive SHR. During development of hypertension both factors get adapted to higher blood pressure level. Sphincter‐like structures in vasa recta suggest contractility of pericytes/vascular smooth muscle cells (vSMC). As these were just seen in SHR that might allude to a higher potential to contract. We conclude that differences in postglomerular structure and function may contribute to the development of hypertension in SHR.

Angiotensin II-NAD(P)H oxidase-stimulated superoxide modifies tubulovascular nitric oxide cross-talk in renal outer medulla.

The source of superoxide (O2*-) production and cell-to-cell interactions of O2*- and nitric oxide (NO) in response to angiotensin II (AngII) were studied by fluorescence microscopic techniques to image rat renal outer medullary microtissue strips. Changes in intracellular O2*- were determined by dihydroethidium-ethidium ratios, and NO was determined with 4,5-diaminofluorescein diacetate. AngII (1 micromol/L) significantly increased O2*- in the isolated, medullary thick ascending limb (mTAL). These responses were inhibited by the superoxide dismutase mimetic 4-hydroxytetramethylpiperidine-1-oxyl (TEMPOL) and by the NAD(P)H oxidase inhibitors diphenylene iodonium and apocynin. AngII did not increase O2*- in either pericytes of isolated, intact vasa recta (VR) or pericytes of VR with a disrupted endothelium, even when surrounded by mTAL. However, AngII did increase O2*- when the tissue strips were preincubated with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), indicating that cross-talk of O2*- from mTAL to the VR occurred but was normally inhibited by NO. Also, tissue O2*- reduction by TEMPOL increased the diffusion of NO from mTAL to the pericytes, indicating that cross-talk of NO from the mTAL to the VR is also inhibited by O2*-. We conclude that AngII stimulates O2*- production in mTAL via the NAD(P)H oxidase pathway and that interactions of O2*- and NO ultimately determine the effectiveness of in situ free-radical cross-talk between the mTAL and the VR.

Lack of UT-B in vasa recta and red blood cells prevents urea-induced improvement of urinary concentrating ability.

Recycling of urea within the renal medulla is known to play an important role in the capacity of the kidney to concentrate urine. This recycling occurs simultaneously through a tubular and a vascular route (i.e., through the loops of Henle and vasa recta, respectively). In the present study, transgenic mice with a selective deficiency in UT-B (the urea transporter protein expressed in descending vasa recta and red blood cells), were used to evaluate the specific contribution of vascular urea recycling to overall urine-concentrating ability (UCA). The renal handling of urea was studied in normal conditions and after acute or chronic alterations in urea excretion (acute urea loading or variations in protein intake, respectively). In normal conditions, UT-B null mice exhibited a 44% elevation in plasma urea (Purea), a normal creatinine clearance, but a 25% decrease in urea clearance, with no change in that of sodium and potassium. Acute urea loading induced a progressive increase in urinary urea concentration (Uurea) in wild-type mice and a subsequent improvement in their UCA in contrast to UT-B null mice, in which urinary osmolality and Uurea did not rise, due to the failure to accumulate urea in the medulla. With increasing protein intake (from 10 to 40% protein in diet, leading to a 5-fold increase in urea excretion), Purea was further increased in null mice while little change was observed in wild-type mice, and null mice were not able to increase Uurea as did wild-type mice. In conclusion, this study in UT-B-deficient mice reveals that countercurrent exchange of urea in renal medullary vessels and red blood cells accounts for a major part of the kidney's concentrating ability and for the adaptation of renal urea handling during a high-protein intake.

Immunolocalization of a microsomal prostaglandin E synthase in rabbit kidney

PGE2, the major cyclooxygenase (COX) metabolite of arachidonic acid, is an important paracrine regulator of numerous tubular and vascular functions in the kidney. To date, COX activity has been considered the key step in prostaglandin synthesis and is well characterized. However, much less is known about the recently cloned microsomal PGE2 synthase (mPGES), the terminal enzyme of PGE2 synthesis, which converts COX-derived PGH2 to the biologically important PGE2. Present studies provide the detailed localization of mPGES protein in the rabbit kidney using immunohistochemistry. In the cortex, strong mPGES labeling was found in the macula densa (MD) and principal cells of the connecting segment and cortical collecting tubule but not in intercalated cells. The medulla was abundant in mPGES-positive structures, with heavy labeling in the collecting duct system. In descending thin limbs and renal medullary interstitial cells, mPGES expression was less intense, and it was below the limits of detection in the vasa recta. Expression of MD mPGES, similarly to COX-2, was greatly increased in response to low-salt diet and angiotensin I-converting enzyme inhibition by captopril. These findings suggest autocrine regulation of renal salt and water transport by PGE2 in descending thin limb and collecting tubule and a paracrine effect of PGE2 on the glomerular and medullary vasculature. Similar to other organs, mPGES in the kidney is an inducible enzyme and may be similarly regulated and acts in concert with COX-2.

The osmotic gradient in kidney medulla: a retold story.

This article is an attempt to simplify lecturing about the osmotic gradient in the kidney medulla. In the model presented, the kidneys are described as a limited space with a positive interstitial hydrostatic pressure. Traffic of water, sodium, and urea is described in levels (or horizons) of different osmolarity, governed by osmotic forces and positive interstitial pressure. In this way, actions of the countercurrent multiplier in nephron tubules and of the countercurrent exchanger in vasa recta are integrated in each horizon. We hope that this approach can help students to better accept conventional presentations in their textbooks.

AQP3, p-AQP2, and AQP2 expression is reduced in polyuric rats with hypercalcemia: prevention by cAMP-PDE inhibitors.

The purpose of this study was to evaluate whether hypercalcemia is associated with downregulation of renal aquaporins (AQPs), including AQP1, AQP2, phosphorylated AQP2 (p-AQP2), AQP3, and AQP4, and if this is the case, to test whether cAMP-phosphodiesterase (PDE) inhibitor treatment can prevent AQP downregulation and prevent the development of polyuria. Vitamin D-induced hypercalcemia in rats was associated with increased urine output and reduced urine osmolality, consistent with previous findings (Levi M, Peterson L, and Berl T. Kidney Int 23: 489-497, 1983). Semiquantitative immunoblotting revealed a significant reduction in the abundance of inner medullary AQP2 (52 +/- 6% of control levels), consistent with previous studies, and of AQP2, which is phosphorylated at the PKA phosphorylation consensus site serine 256 (p-AQP2; 36 +/- 8%). Moreover, AQP3 abundance was also significantly decreased (45 +/- 7 and 61 +/- 6% of control levels in inner medulla and whole kidney, respectively). Consistent with this, immunohistochemistry demonstrated reduced AQP3 immunolabeling along the entire collecting duct. AQP4 expression was not reduced. Surprisingly, total kidney AQP1 abundance was also reduced (60 +/- 6%). AQP1 expression was reduced in the cortex and outer stripe of the outer medulla (48 +/- 7%; i.e., in proximal tubules). In contrast, AQP1 levels were not changed in the inner stripe of the outer medulla or in the inner medulla (i.e., descending thin limbs and vasa recta). Treatment with the cAMP-PDE inhibitors rolipram and milrinone in combination (inhibiting PDE IV and PDE III isoenzymes) at day 2 and onward completely prevented the hypercalcemia-induced downregulation of AQP2 and AQP3 (but not AQP1) and completely prevented the development of polyuria. In conclusion, AQP3, AQP2, and p-AQP2 are downregulated and are likely to play critical roles in the development of polyuria associated with vitamin D-induced hypercalcemia. Moreover, PDE inhibitor treatment significantly prevented the reduced expression of collecting duct AQPs and prevented the development of polyuria.

P-selectin–dependent macrophage migration into the tubulointerstitium in unilateral ureteral obstruction

Interstitial infiltration of macrophages (Mø) is one of the main causal factors for the tubulointerstitial injury. However, precise mechanisms of Mø infiltration into tubulointerstitium have not been fully explored. The purposes of this study were to assess the role of selectins in the acute infiltration of Mø in rats with unilateral ureteral obstruction (UUO) and to evaluate the role of vasa recta, that is, whether they facilitate massive influx of Mø into the interstitium by functioning as specialized vessels. To evaluate the role of selectins in Mø infiltration into tubulointerstitium, the expression of selectins and L-selectin ligands was examined by immunohistochemistry and immunoelectron microscopy. The functional role of P-selectin in vasa recta was studied by Stamper-Woodruff assay, in vivo p-Mø migration assay and in vivo blocking experiments with the monoclonal antibody (mAb) ARP2-4. Selective expression of P-selectin was detected in vasa recta as early as one hour after UUO, and the expression increased thereafter for 96 hours. In contrast, endothelial expression of L-selectin ligands and E-selectin were not detectable. In the Stamper-Woodruff assay on kidney sections of rats with UUO, the adhesion of isolated rat peritoneal Mø (p-Mø) to vasa recta was significantly inhibited by the mAb ARP2-4 (P-selectin blocker; P < 0.01), but not by mAb ARE-5 (E-selectin blocker) or rLECIg (rat L-selectin chimeric protein). In the in vivo transfer experiments with fluorescein-labeled p-Mø into rats 48 hours after UUO, labeled p-Mø had accumulated around vasa recta at three minutes and had infiltrated predominantly into the outer medulla at 180 minutes. The number of labeled p-Mø was reduced when the rats were pretreated with ARP2-4 (P < 0.01). Finally, ARP2-4 (10 mg/kg), injected 15 minutes before UUO, reduced the number of infiltrated Mø (P < 0.01). The results suggest that vasa recta, which express P-selectin, contribute to massive infiltration of Mø into the interstitium by functioning as specialized post-capillary venules.

Peroxisome Proliferator-activated Receptor δ Activation Promotes Cell Survival following Hypertonic Stress

COX2-selective non-steroidal anti-inflammatory drugs (NSAIDs) cause selective apoptosis of renal medullary interstitial cells (RMIC) in vivo and reduce their ability to tolerate hypertonic stress in vitro. To determine the mechanism by which COX2 activity promotes RMIC viability, we examined the capacity of COX2-derived prostanoids to promote RMIC survival. Although RMICs synthesize prostaglandin E2 (PGE2) PGI2 > PGF2a > TxA2, only PGI2 enhanced RMIC viability following hypertonic stress. RMICs do not express the prostacyclin receptor, but they do express the prostacyclin responsive nuclear transcription factor peroxisome proliferator-activated receptor delta (PPARdelta). Hypertonic stress increased PGI2 synthesis 330% above base line and also activated a PPARdelta specific reporter (delta response element (DRE)) by 90% above base line. Conversely DRE activity was only inhibited by the COX2-selective inhibitor SC236 but not by a COX1-selective NSAID (SC560). Overexpression of PPARdelta using an adenovirus not only drove DRE activity but also prevented RMIC death due to COX2 inhibition. These studies are consistent with a model whereby hypertonicity activates COX2-derived prostaglandin production, which promotes RMIC viability through PPARdelta. Inhibition of PPARdelta activity may contribute to the renal papillary necrosis associated with analgesic and/or NSAID use.

Role of aquaporins in endothelial water transport

The aquaporins (AQP) are a family of homologous water channels expressed in many epithelial and endothelial cell types involved in fluid transport. AQP1 protein is strongly expressed in most microvascular endothelia outside of the brain as well as in endothelial cells in cornea, intestinal lacteals, and other tissues. AQP4 is expressed in astroglial foot processes adjacent to endothelial cells in the central nervous system. Transgenic mice lacking aquaporins have been useful in defining their role in mammalian physiology. Mice lacking AQP1 manifest defective urinary concentrating ability, in part because of decreased water permeability in renal vasa recta microvessels. These mice also show a defect in dietary fat processing that may involve chylomicron absorption by intestinal lacteals. There is preliminary evidence that AQP1 might play a role in tumour angiogenesis and in renal microvessel structural adaptation. However AQP1 in most endothelial tissues does not appear to have a physiological function despite its role in osmotically driven water transport. For example mice lacking AQP1 have low alveolar capillary water permeability but unimpaired lung fluid absorption, as well as unimpaired saliva and tear secretion, aqueous fluid outflow, and pleural and peritoneal fluid transport. In the central nervous system mice lacking AQP4 are partially protected from brain oedema in water intoxication and ischaemic models of brain injury. Therefore although the role of aquaporins in epithelial fluid transport is in most cases well understood there remain many questions about the role of aquaporins in endothelial cell function. It is unclear why many leaky microvessels strongly express AQP1 without apparent functional significance. Improved understanding of aquaporin endothelial biology may lead to novel therapies for human disease, such as pharmacological modulation of tumour angiogenesis, renal fluid clearance and intestinal absorption.

Aquaporin gene delivery to kidney

Several aquaporin- (AQP) type water channels are expressed in kidney tubules and microvessels, including AQP1 in proximal tubule, thin descending limb of Henle and vasa recta, AQP2 in collecting duct apical membrane, and AQP3 and AQP4 in collecting duct basolateral membrane. Mice deficient in these aquaporins have distinct phenotypic abnormalities. AQP1 null mice are polyuria and unable to generate a concentrated urine after water deprivation. AQP2-T126M mutant mice and AQP3 null mice manifest nephrogenic diabetes insipidus (NDI) with severe polyuria, whereas AQP4 null mice have only a mild defect in maximal urinary concentrating ability. We reasoned that these mice could serve as useful models for gene replacement because of their predictable and unambiguous phenotypes. In an initial feasibility study, an adenovirus directing the expression of AQP1 was introduced into AQP1 null mice by intravenous infusion. At 1 week after adenovirus infusion, AQP1 was seen in many proximal tubules and microvessels. Compared with untreated null mice, the treated mice were able to partially concentrate their urine and lost less weight after water deprivation. However, AQP1 transgene expression and functional correction were lost over 3-5 weeks. Although there remain many technical problems to overcome, aquaporin gene replacement has potential applications in hereditary and acquired NDI, and in the transient modulation of renal fluid conservation.

Drainage of plasma proteins from the renal medullary interstitium in rats.

1. Lymph vessels are scarce or lacking in the renal inner medulla, raising the question of whether plasma proteins entering the medullary interstitium are removed by diffusion through the interstitium to lymphatics in the outer medulla or cortex, or by convection into the vasa recta. 2. Using micropipettes, we infused 125I-albumin into the papilla of anaesthetized rats and watched its disappearance from the injection site as well as the uptake in the thoracic duct and plasma. 3. Tracer infused into the renal cortex appeared almost immediately in the thoracic duct lymph, and rose to a sevenfold higher concentration than in plasma, whereas tracer infused into the papilla appeared first and increased more sharply in plasma than in the lymph. No spread from the papillary injection site was observed. Tracer injected in renal hilar lymphatics was quantitatively recovered in the thoracic duct. 4. The plasma concentration pattern following papillary infusion was similar to that obtained by intravenous injection, indicating uptake in blood and subsequent distribution to extracellular fluid and lymph from all organs. 5. We conclude that plasma proteins normally diffusing out from the vasa recta are brought back through water flux (1) from the collecting ducts due to the high sodium chloride concentration in the papillary interstitium and (2) from the interstitium into the vasa recta driven by plasma protein osmotic pressure. Accordingly, there is no need for lymph vessels in the inner medulla.

Role of aquaporin water channels in kidney function studied using transgenic mice

Several aquaporin (AQP) water transporting proteins are expressed in mammalian kidney: AQP1 in plasma membranes of proximal tubule, thin descending limb of Henle, and descending vasa recta; AQP2 in collecting duct luminal membrane; AQP3 and AQP4 in collecting duct basolateral membrane; AQP6 in intercalated cells; and AQP7 in the S3 segment of proximal tubule. To define the role of aquaporins in renal physiology, we have generated and characterized transgenic null mice deficient in AQP1, AQP3, and AQP4, individually and in combinations, as well as AQP2 mutant mice, in which the T126M mutation causing human nephrogenic diabetes insipidus was introduced. AQP1-deficient mice are polyuric and unable to concentrate their urine in response to water deprivation or vasopressin administration. AQP1 deletion greatly reduces osmotic water permeability in proximal tubule, thin descending limb of Henle, and descending vasa recta, resulting in defective proximal tubule fluid absorption and medullary countercurrent exchange. Mice lacking AQP3 have low basolateral membrane water permeability in cortical collecting duct and excrete large quantities of dilute urine. Mice lacking AQP4 have low water permeability in inner medullary collecting duct, but manifest only a mild defect in maximum urinary concentrating ability. These data, taken together with phenotype analyses of brain, lung, and gastrointestinal organs, support the paradigm that aquaporins facilitate rapid near-isosmolar transepithelial fluid absorption/secretion, as well as rapid vectorial water movement driven by osmotic gradients. The renal phenotype data in aquaporin knockout mice suggests the utility of aquaporin blockers as novel aquaretic-diuretic agents.

Vascular smooth muscle cells express the alpha(1A) subunit of a P-/Q-type voltage-dependent Ca(2+)Channel, and It is functionally important in renal afferent arterioles.

In the present study, we tested whether the alpha(1A) subunit, which encodes a neuronal isoform of voltage-dependent Ca(2+) channels (VDCCs) (P-/Q-type), was present and functional in vascular smooth muscle and renal resistance vessels. By reverse transcription-polymerase chain reaction and Southern blotting analysis, mRNA encoding the alpha(1A) subunit was detected in microdissected rat preglomerular vessels and vasa recta, in cultures of rat preglomerular vascular smooth muscle cells (VSMCs), and in cultured rat mesangial cells. With immunoblots, alpha(1A) subunit protein was demonstrated in rat aorta, brain, aortic smooth muscle cells (A7r5), VSMCs, and mesangial cells. Immunolabeling with an anti-alpha(1A) antibody was positive in acid-macerated, microdissected preglomerular vessels and in A7r5 cells. Patch-clamp experiments on aortic A7r5 cells showed 22+/-4% (n=6) inhibition of inward Ca(2+) current by omega-Agatoxin IVA (10(-8) mol/L), which in this concentration is a specific inhibitor of P-type VDCCs. Measurements of intracellular Ca(2+) in afferent arterioles with fluorescence-imaging microscopy showed 32+/-9% (n=10) inhibition of the K(+)-induced rise in Ca(2+) in the presence of 10(-8) mol/L omega-Agatoxin IVA. In microperfused rabbit afferent arterioles, omega-Agatoxin IVA inhibited depolarization-mediated contraction with an EC(50) of 10(-17) mol/L and complete blockade at 10(-14) mol/L. We conclude that the alpha(1A) subunit is expressed in VSMCs from renal preglomerular resistance vessels and aorta, as well as mesangial cells, and that P-type VDCCs contribute to Ca(2+) influx in aortic and renal VSMCs and are involved in depolarization-mediated contraction in renal afferent arterioles.

CD59 protects rat kidney from complement mediated injury in collaboration with Crry

As previously reported, the membrane-bound complement regulator at the C3 level (Crry/p65) is important in maintaining normal integrity of the kidney in rats. However, the role of a complement regulator at the C8/9 level (CD59) is not clear, especially when activation of complement occurs at the C3 level. The aim of this work was to elucidate the in vivo role of CD59 under C3 activating conditions. Two monoclonal antibodies, 5I2 and 6D1, were used to suppress the function of Crry and CD59, respectively. In order to activate alternative the pathway of complement, the left kidney was perfused with 5I2 and/or 6D1 and was recirculated. In the kidneys perfused with 5I2 alone, deposition of C3 and membrane attack complex (MAC) was observed in the peritubular capillaries, vasa recta, and tubular basement membranes. Cast formation, tubular dilation and degeneration, and cellular infiltration were observed at days 1 and 4, and they recovered by day 7. Further suppression of CD59 by 6D1 significantly enhanced the deposition of MAC and worsened the already exacerbated tubulointerstitial injury. These effects of 6D1 were dose dependent. Perfusion with 6D1 alone did not induce histologic damage or MAC deposition in the tubulointerstitium. In rats, CD59 maintains normal integrity of the kidney in collaboration with Crry in rats against complement-mediated damage in vivo.

Physiological importance of aquaporins: lessons from knockout mice.

The phenotype analysis of transgenic mice deficient in specific aquaporin water channels has provided new insights into the role of aquaporins in organ physiology. AQP1-deficient mice are polyuric and are unable to concentrate their urine in response to water deprivation or vasopressin administration. AQP1 deletion reduces osmotic water permeability in the proximal tubule, thin descending limb of Henle and vasa recta, resulting in defective proximal tubule fluid absorption and medullary countercurrent exchange. Mice lacking AQP3, a basolateral membrane water channel expressed mainly in the cortical collecting duct, are remarkably polyuric but are able to generate a partly concentrated urine after water deprivation. In contrast, mice lacking AQP4, a water channel expressed mainly in the inner medullary collecting duct, manifest only a mild defect in maximum urinary concentrating ability. These data, together with phenotype analyses of the brain, lung, salivary gland, and gastrointestinal organs, support the paradigm that aquaporins can facilitate near-isosmolar transepithelial fluid absorption/secretion as well as rapid vectorial water movement driven by osmotic gradients. The phenotype data obtained from aquaporin knockout mice suggest the utility of aquaporin blockers as novel diuretic agents.

Inner medullary lactate production and accumulation: a vasa recta model

Since anaerobic glycolysis yields two lactates for each glucose consumed and since it is reported to be a major source of ATP for inner medullary (IM) cell maintenance, it is a likely source of "external" IM osmoles. It has long been known that such an osmole source could theoretically contribute to the "single-effect" of the urine concentrating mechanism, but there was previously no suggestion of a plausible source. I used numerical simulation to estimate axial gradients of lactate and glucose that might be accumulated by countercurrent recycling in IM vasa recta (IMVR). Based on measurements in other tissues, anaerobic glycolysis (assumed to be independent of diuretic state) was estimated to consume approximately 20% of the glucose delivered to the IM. IM tissue mass and axial distribution of loops and vasa recta were according to reported values for rat and other rodents. Lactate (P(LAC)) and glucose (P(GLU)) permeabilities were varied over a range of plausible values. The model results suggest that P(LAC) of 100 x 10(-5) cm/s (similar to measured permeabilities for other small solutes) is sufficiently high to ensure efficient lactate recycling. By contrast, it was necessary in the model to reduce P(GLU) to a small fraction of this value (1/25th) to avoid papillary glucose depletion by countercurrent shunting. The results predict that IM lactate production could suffice to build a significant steady-state axial lactate gradient in the IM interstitium. Other modeling studies (Jen JF and Stephenson JL. Bull Math Biol 56: 491-514, 1994; and Thomas SR and Wexler AS. Am J Physiol Renal Fluid Electrolyte Physiol 269: F159-F171, 1995) have shown that 20-100 mosmol/kgH(2)O of unspecified external, interstitial, osmolytes could greatly improve IM concentrating ability. The present study gives several plausible scenarios consistent with accumulation of metabolically produced lactate osmoles, although only to the lower end of this range. For example, if 20% of entering glucose is consumed, the model predicts that papillary lactate would attain about 15 mM assuming vasa recta outflow is increased 30% by fluid absorbed from the nephrons and collecting ducts and that this lactate gradient would double if IM blood flow were reduced by one-half, as may occur in antidiuresis. Several experimental tests of the hypothesis are indicated.

Formation and Actions of Cyclic ADP-Ribose in Renal Microvessels

Recent studies indicated that cyclic ADP-ribose (cADPR) serves as a second messenger for intracellular Ca2+ mobilization in a variety of mammalian cells. However, the metabolism and actions of cADPR in the renal vasculature are poorly understood. In the present study, we characterized the enzymatic pathway of the production and metabolism of cADPR along the renal vascular tree and determined the role of cADPR in the control of intracellular [Ca2+] and vascular tone. The high performance liquid chromatographic analyses showed that cADPR was produced and hydrolyzed along the renal vasculature. The maximal conversion rate of nicotinamide guanine dinucleotide (NGD) into cyclic GDP-ribose (that represents ADP-ribosyl cyclase activity for cADPR formation) was 8.69 ± 2.39 nmol/min/mg protein in bulk-dissected intrarenal preglomerular vessels (n = 7) and 4.35 ± 0.13, 2.23 ± 0.27, 2.40 ± 0.19, and 0.31 ± 0.02 nmol/min/mg protein, respectively, in microdissected arcuate arteries (n = 6), interlobular arteries (n = 6), afferent arterioles (n = 7), and vasa recta (n = 10). The activity of cADPR hydrolase was also detected in the renal vasculature. Using the fluorescence microscopic spectrometry, cADPR was found to produce a large rapid Ca2+ release from β-escin-permeabilized renal arterial smooth muscle cells (SMCs). In isolated, perfused, and pressurized small renal arteries, cADPR produced a concentration-dependent vasoconstriction when added into the bath solution. The vasoconstrictor effect of cADPR was completely blocked by tetracaine, a Ca2+-induced Ca2+ release (CICR) inhibitor. These results suggest that an enzymatic pathway for cADPR production and metabolism is present along the renal vasculature and that cADPR may importantly contribute to the control of renal vascular tone through CICR.

Intracellular pH in isolated rat renal papillary thin limbs of Henle's loop.

Intracellular pH (pHi) was measured in isolated, nonperfused and perfused rat papillary thin limbs of Henle's loops in N-2-hydroxyethylpiperazine-N'-2-ethansulfonic acid (HEPES)- or HEPES/bicarbonate-buffered medium at pH 7.4 using the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). Resting pHi was about 6.7 in descending thin limbs (DTL) and about 6.9 in ascending thin limbs (ATL), even with a medium pH of 7.4. These values appeared to reflect the acid pH of the blood in the neighboring vasa recta found in vivo. The resting pHi did not differ whether or not the medium contained bicarbonate although the total buffering capacity of the tubule cells was increased in the presence of bicarbonate. In nonperfused DTL and ATL, pHi was further acidified following an NH4Cl pulse. The rate of recovery of pHi from this level to the resting pHi was reduced by Na+ removal from the bath in both DTL and ATL and by the addition of ethylisopropylamiloride (EIPA) to the bath in the presence of Na+ in DTL. The rate of recovery was not affected by Cl- removal from the bath or K+ (75 mM) or 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) addition to the bath in either DTL or ATL. These results suggest that the common, amiloride-sensitive, basolateral Na+/H+ exchanger plays a role in the regulation of pHi in rat papillary DTL but that a different basolateral Na+/H+ exchanger or a luminal Na+/H+ exchanger is important in rat papillary ATL.

Cycloleucine fluxes during rat vasa recta and loop microinfusions in vivo and loop microperfusions in vitro.

Amino acids are apparently recycled between loops of Henle and vasa recta in rat papilla in vivo. To examine this process in the absence of metabolism, we performed continuous microinfusions of rat renal papillary ascending thin limbs (ATLs) and vasa recta in vivo, and microperflusions of isolated rat renal papillary descending thin limbs (DTLs) and ATLs in vitro using the nonmetabolizable, synthetic, neutral amino acid cycloleucine. Like naturally occurring amino acids, approximately = 25% of radiolabeled cycloleucine microinfused into ATLs in vivo was reabsorbed by a process that was not saturable or inhibitable. Also, like naturally occurring amino acids, approximately = 47% (relative to inulin) of radiolabeled cycloleucine microinfused into ascending vasa recta in vivo was transferred directly into ipsilateral tubular structures (probably DTLs) by a saturable and inhibitable process. In DTLs perfused in vitro, unidirectional bath-to-lumen fluxes (Jbl) tended to exceed unidirectional lumen-to-bath fluxes (Jlb), whereas in ATLs perfused in vitro Jlb tended to exceed Jbl, but the differences were not statistically significant. Moreover, none of the unidirectional fluxes was saturable or inhibitable, an observation compatible with apparent reabsorption from ATLs in vivo but incompatible with apparent movement from vasa recta to DTLs in vivo. These in vitro observations are like those made previously for the naturally occurring neutral amino acid L-alanine. The lack of saturation and inhibition, like the previous data on L-alanine, suggest that transepithelial movement of amino acids in thin limbs of Henle's loop may occur via a paracellular route and that regulation of amino acid movement in vivo may involve vasa recta, not DTLs. They also suggest that cycloleucine is a good nonmetabolizable surrogate for the study of neutral amino acid transport in the kidney.