ATP mediates flow-induced NO production in thick ascending limbs

Mechanical stimulation caused by increasing flow induces nucleotide release from many cells. Luminal flow and extracellular ATP stimulate production of nitric oxide (NO) in thick ascending limbs. However, the factors that mediate flow-induced NO production are unknown. We hypothesized that luminal flow stimulates thick ascending limb NO production via ATP. We measured NO in isolated, perfused rat thick ascending limbs using the fluorescent dye DAF FM. The rate of increase in dye fluorescence reflects NO accumulation. Increasing luminal flow from 0 to 20 nl/min stimulated NO production from 17 ± 16 to 130 ± 37 arbitrary units (AU)/min (P < 0.02). Increasing flow from 0 to 20 nl/min raised ATP release from 4 ± 1 to 21 ± 6 AU/min (P < 0.04). Hexokinase (10 U/ml) plus glucose, which consumes ATP, completely prevented the measured increase in ATP. Luminal flow did not increase NO production in the presence of luminal and basolateral hexokinase (10 U/ml). When flow was increased with the ATPase apyrase in both luminal and basolateral solutions (5 U/ml), NO levels did not change significantly. The P2 receptor antagonist suramin (300 μmol/l) reduced flow-induced NO production by 83 ± 25% (P < 0.03) when added to both and basolateral sides. Luminal hexokinase decreased flow-induced NO production from 205.6 ± 85.6 to 36.6 ± 118.6 AU/min (P < 0.02). Basolateral hexokinase also reduced flow-induced NO production. The P2X receptor-selective antagonist NF023 (200 μmol/l) prevented flow-induced NO production when added to the basolateral side but not the luminal side. We conclude that ATP mediates flow-induced NO production in the thick ascending limb likely via activation of P2Y receptors in the luminal and P2X receptors in the basolateral membrane.     

Mathematical model of nitric oxide convection and diffusion in a renal medullary vas rectum

In this study, the generation, convection, diffusion, and consumption of nitric oxide (NO) in and around a single renal medullary descending or ascending vas rectum in rat were modeled using CFD. The vascular lumen (with a core RBC-rich layer and a parietal layer), the endothelium, the pericytes and the interstitium were represented as concentric cylinders. We accounted for the generation of NO by vascular endothelial cells, and that by the epithelial cells of medullary thick ascending limbs (mTALs) and inner medullary collecting ducts (IMCDs), the latter via interstitial boundary conditions. Luminal velocity profiles were obtained by modeling blood flow dynamics. Our results suggest that convection (i.e., blood flow per se) does not significantly affect NO concentrations along the cortico–medullary axis, because the latter are mostly determined by the rate of NO production and that of NO consumption by hemoglobin. However, the shear stress-mediated effects of blood flow on NO generation rates, and therefore NO concentrations, were predicted to be important. Finally, we found that unless epithelial NO generation rates (per unit tubular surface area) are at least 10 times lower than endothelium NO generation rates, NO production by mTALs and IMCDs affects vascular NO concentrations, with possible consequences for medullary blood flow distribution.     

Guanylyl cyclase-C receptor mRNA distribution along the rat nephron

Guanylin (GN) and uroguanylin (UGN) are two recently identified peptides that have been shown to affect water and electrolyte transport in both the intestine and the kidney. Mechanistically, the effects of both peptides are thought to be mediated by intracellular cGMP which results from ligand binding to a plasma membrane guanylyl cyclase-C (GC-C) receptor. To date, the specific intrarenal site(s) of GN and UGN action have not been established. To begin to address this issue, the present studies utilized semi-quantitative RT-PCR to assess the distribution of GC-C mRNA in specific microdissected segments of the rat nephron. GC-C mRNA expression was highest in the cortical collecting tubule, followed by the proximal convoluted tubule, medullary thick ascending limb and collecting tubule, and thin limbs of Henle's loop. Expression levels were significantly lower in all other segments tested, including the glomerulus. The renal tubular expression pattern for cGMP-dependent protein kinase II (cGK-II) mRNA, which is activated in response to GN/UGN-dependent cGMP accumulation, was similar to that for GC-C. Notably, both GN and UGN mRNAs were also expressed along the nephron. The highest levels of expression for both peptides were detected in the medullary collecting tubule. Lower, but comparable levels of GN and UGN expression also occurred in the cortical collecting tubule, cortical and medullary thick ascending limb, and thin limbs of Henles loop. In the proximal convoluted tubule, GN mRNA expression was also quite high, while UGN mRNA was almost undetectable. The presence of renal GC-C and cGK-II in the kidney are consistent with a proposed endocrine function for GN and UGN. In addition however, the present data suggest that intrarenally synthesized GN and UGN may also contribute to the regulation of renal tubular transport.     

Effects of low-potassium diet on N-ethylmaleimide-sensitive ATPase in the distal nephron segments.

The present study was undertaken to investigate whether or not potassium deficiency influences N-ethylmaleimide (NEM)-sensitive ATPase in the distal nephron segments of the rat. One group of animals was fed a low-K diet, whereas the normal K-group was given the same diet after supplementation with KCl. The nephron segments examined were: the medullary and cortical thick ascending limbs, the distal convoluted tubule, and the cortical, outer and inner medullary collecting ducts. NEM-sensitive ATPase activity in microdissected segments was measured by a fluorometric microassay. The plasma K+ concentration in the low-K group was 3.1 +/- 0.3 mEq/l compared with 4.2 +/- 0.1 mEq/l in the normal-K group. NEM-sensitive ATPase activity in the outer medullary collecting duct of low-K diet animals was significantly greater than in normal-K animals. There was no significant difference in NEM-sensitive ATPase activity between the two groups of animals in the other nephron segments examined. It is suggested that NEM-sensitive H-ATPase activity in the outer medullary collecting duct is modulated by the potassium status of the animal.     

Benzodiazepine receptors along the nephron: [3H]PK 11195 binding in rat tubules

Binding of [3H]PK 11195, an isoquinoline carboxamide derivative, was measured in microdissected tubule segments of rat nephron. High specific binding capacities (1.1-1.8 fmol X mm-1) were found in the thick ascending limb of the Henle's loop and in the collecting tubule, whereas specific binding could not be detected in the proximal tubule. In the medullary collecting tubule, the association and dissociation rate constants at 4 degrees C were k1 = 3.0 X 10(6) M-1 X min-1 and k-1 = 0.021 min -1; the ratio k-1/k1 = 7.0 nM was in agreement with the estimated equilibrium dissociation constant (Kd = 2.4 nM). [3H]PK 11195 binding sites from medullary ascending limb and medullary collecting tubule revealed the following sequence of specificity: PK 11195 = Ro 5-4864 much greater than clonazepam, indicating that tubule binding sites might be the peripheral benzodiazepine receptors of the rat kidney.     

Modulation of outer medullary NaCl transport and oxygenation by nitric oxide and superoxide

We expanded our region-based model of water and solute exchanges in the rat outer medulla to incorporate the transport of nitric oxide (NO) and superoxide (O(2)(-)) and to examine the impact of NO-O(2)(-) interactions on medullary thick ascending limb (mTAL) NaCl reabsorption and oxygen (O(2)) consumption, under both physiological and pathological conditions. Our results suggest that NaCl transport and the concentrating capacity of the outer medulla are substantially modulated by basal levels of NO and O(2)(-). Moreover, the effect of each solute on NaCl reabsorption cannot be considered in isolation, given the feedback loops resulting from three-way interactions between O(2), NO, and O(2)(-). Notwithstanding vasoactive effects, our model predicts that in the absence of O(2)(-)-mediated stimulation of NaCl active transport, the outer medullary concentrating capacity (evaluated as the collecting duct fluid osmolality at the outer-inner medullary junction) would be ～40% lower. Conversely, without NO-induced inhibition of NaCl active transport, the outer medullary concentrating capacity would increase by ～70%, but only if that anaerobic metabolism can provide up to half the maximal energy requirements of the outer medulla. The model suggests that in addition to scavenging NO, O(2)(-) modulates NO levels indirectly via its stimulation of mTAL metabolism, leading to reduction of O(2) as a substrate for NO. When O(2)(-) levels are raised 10-fold, as in hypertensive animals, mTAL NaCl reabsorption is significantly enhanced, even as the inefficient use of O(2) exacerbates hypoxia in the outer medulla. Conversely, an increase in tubular and vascular flows is predicted to substantially reduce mTAL NaCl reabsorption. In conclusion, our model suggests that the complex interactions between NO, O(2)(-), and O(2) significantly impact the O(2) balance and NaCl reabsorption in the outer medulla.     

Transendothelial flow inhibits neutrophil transmigration through a nitric oxide-dependent mechanism: potential role for cleft shear stress

Endothelial cells in vivo are well known to respond to parallel shear stress induced by luminal blood flow. In addition, fluid filtration across endothelium (transendothelial flow) may trigger nitric oxide (NO) production, presumably via shear stress within intercellular clefts. Since NO regulates neutrophil-endothelial interactions, we determined whether transendothelial flow regulates neutrophil transmigration. Interleukin-1beta-treated human umbilical vein endothelial cell (HUVEC) monolayers cultured on a polycarbonate filter were placed in a custom chamber with or without a modest hydrostatic pressure gradient (DeltaP, 10 cm H(2)O) to induce transendothelial flow. In other experiments, cells were studied in a parallel plate flow chamber at various transendothelial flows (DeltaP = 0, 5, and 10 cm H(2)O) and luminal flows (shear stress of 0, 1, and 2 dyn/cm(2)). In the absence of luminal flow, transendothelial flow reduced transmigration of freshly isolated human neutrophils from 57% to 14% (P < 0.05) and induced an increase in NO detected with a fluorescent assay (DAF-2DA). The NO synthase inhibitor L-NAME prevented the effects of transendothelial flow on neutrophil transmigration, while a NO donor (DETA/NO, 1 mM) inhibited neutrophil transmigration. Finally, in the presence of luminal flow (1 and 2 dyn/cm(2)), transendothelial flow also inhibited transmigration. On the basis of HUVEC morphometry and measured transendothelial volume flow, we estimated cleft shear stress to range from 49 to 198 dyn/cm(2). These shear stress estimates, while substantial, are of similar magnitude to those reported by others with similar analyses. These data are consistent with the hypothesis that endothelial cleft shear stress inhibits neutrophil transmigration via a NO-dependent mechanism.     

A Na+- and Cl- -activated K+ channel in the thick ascending limb of mouse kidney

This study investigates the presence and properties of Na+-activated K+ (K(Na)) channels in epithelial renal cells. Using real-time PCR on mouse microdissected nephron segments, we show that Slo2.2 mRNA, which encodes for the K(Na) channels of excitable cells, is expressed in the medullary and cortical thick ascending limbs of Henle's loop, but not in the other parts of the nephron. Patch-clamp analysis revealed the presence of a high conductance K+ channel in the basolateral membrane of both the medullary and cortical thick ascending limbs. This channel was highly K+ selective (P(K)/P(Na) approximately 20), its conductance ranged from 140 to 180 pS with subconductance levels, and its current/voltage relationship displayed intermediate, Na+-dependent, inward rectification. Internal Na+ and Cl- activated the channel with 50% effective concentrations (EC50) and Hill coefficients (nH) of 30 +/- 1 mM and 3.9 +/- 0.5 for internal Na+, and 35 +/- 10 mM and 1.3 +/- 0.25 for internal Cl-. Channel activity was unaltered by internal ATP (2 mM) and by internal pH, but clearly decreased when internal free Ca2+ concentration increased. This is the first demonstration of the presence in the epithelial cell membrane of a functional, Na+-activated, large-conductance K+ channel that closely resembles native K(Na) channels of excitable cells. This Slo2.2 type, Na+- and Cl--activated K+ channel is primarily located in the thick ascending limb, a major renal site of transcellular NaCl reabsorption.     

Flash photolysis of caged nitric oxide inhibits proximal tubular fluid reabsorption in free-flow nephron

A nonobstructing optical method was developed to measure proximal tubular fluid reabsorption in rat nephron at 0.25 Hz. The effects of uncaging luminal nitric oxide (NO) on proximal tubular reabsorption were investigated with this method. Proximal fluid reabsorption rate was calculated as the difference of tubular flow measured simultaneously at two locations (0.8-1.8 mm apart) along a convoluted proximal tubule. Tubular flow was estimated on the basis of the propagating velocity of fluorescent dextran pulses in the lumen. Changes in local tubular flow induced by intratubular perfusion were detected simultaneously along the proximal tubule, indicating that local tubular flow can be monitored in multiple sites along a tubule. The estimated tubular reabsorption rate was 5.52 +/- 0.38 nl.min(-1).mm(-1) (n = 20). Flash photolysis of luminal caged NO (potassium nitrosylpentachlororuthenate) was induced with a 30-Hz UV nitrogen-pulsed laser. Release of NO from caged NO into the proximal tubule was confirmed by monitoring intracellular NO concentration using a cell-permeant NO-sensitive fluorescent dye (DAF-FM). Emission of DAF-FM was proportional to the number of laser pulses used for uncaging. Photolysis of luminal caged NO induced a dose-dependent inhibition of proximal tubular reabsorption without activating tubuloglomerular feedback, whereas uncaging of intracellular cGMP in the proximal tubule decreased tubular flow. Coupling of this novel method to measure reabsorption with photolysis of caged signaling molecules provides a new paradigm to study tubular reabsorption with ambient tubular flow.     

Aging and estrogen alter endothelial reactivity to reactive oxygen species in coronary arterioles

Endothelium-dependent, nitric oxide (NO)-mediated vasodilation can be impaired by reactive oxygen species (ROS), and this deleterious effect of ROS on NO availability may increase with aging. Endothelial function declines rapidly after menopause, possibly because of loss of circulating estrogen and its antioxidant effects. The purpose of the current study was to determine the role of O(2)(-) and H(2)O(2) in regulating flow-induced dilation in coronary arterioles of young (6-mo) and aged (24-mo) intact, ovariectomized (OVX), or OVX + estrogen-treated (OVE) female Fischer 344 rats. Both aging and OVX reduced flow-induced NO production, whereas flow-induced H(2)O(2) production was not altered by age or estrogen status. Flow-induced vasodilation was evaluated before and after treatment with the superoxide dismutase (SOD) mimetic Tempol (100 μM) or the H(2)O(2) scavenger catalase (100 U/ml). Removal of H(2)O(2) with catalase reduced flow-induced dilation in all groups, whereas Tempol diminished vasodilation in intact and OVE, but not OVX, rats. Immunoblot analysis revealed elevated nitrotyrosine with aging and OVX. In young rats, OVX reduced SOD protein while OVE increased SOD in aged rats; catalase protein did not differ in any group. Collectively, these studies suggest that O(2)(-) and H(2)O(2) are critical components of flow-induced vasodilation in coronary arterioles from female rats; however, a chronic deficiency of O(2)(-) buffering by SOD contributes to impaired flow-induced dilation with aging and loss of estrogen. Furthermore, these data indicate that estrogen replacement restores O(2)(-) homeostasis and flow-induced dilation of coronary arterioles, even at an advanced age.     

Glycocalyx heterogeneity of rat kidney urinary tubule: demonstration with a lectin-gold technique specific for sialic acid

The distribution of sialic acid residues in rat kidney urinary tubule was investigated by light and electron microscopy with a lectin-gold technique. The application of the sialic acid specific Limax flavus lectin resulted in intense plasma membrane labeling of the epithelium of the entire proximal tubule and thin limbs of loop of Henle. In contrast, the plasma membrane of the epithelium lining the medullary portion of the thick ascending limb of Henle was not labeled. In the cortical portion, however, microvilli-bearing positive and smooth-surfaced negative cells were present. Moreover, all cells of the convoluted distal tubules were labeled along their plasma membrane. These data demonstrate the existence of a gross difference in glycocalyx composition between proximal tubules and thin limbs of loop of Henle on one hand and thick ascending limbs on the other. In addition, fine heterogeneity in glycocalyx composition between medullary and cortical portion of thick ascending limb exists. It is concluded that the differences in sialic acid content of the glycocalyx may be related to the functional diversity exhibited by these tubular regions.     

Cellular localization of THIK-1 (K(2P)13.1) and THIK-2 (K(2P)12.1) K channels in the mammalian kidney.

K(+)-channels fulfill several important functions in the mammalian kidney such as volume regulation, recirculation and secretion of K(+) ions, and maintaining the resting potential. In this study we used immunocytochemical methods, in situ hybridization, and nephron segment-specific RT-PCR to obtain a detailed picture of the cellular localization of two tandem pore domain potassium (K(2P)) channels, THIK-1 (K(2P)13.1, KCNK13) and THIK-2 (K(2P)12.1, KCNK12). Monospecific antibodies against C-terminal domains of rat THIK-1 and THIK-2 proteins (GST-fusion proteins) were raised in rabbits, freed from cross-reactivity, and affinity purified. All antibodies were validated by Western blot analysis, competitive ELISA, and preabsorption experiments. The expression of THIK channels in specific nephron segments was confirmed by double staining with marker proteins. Results indicate that in rat and mouse THIK-1 and THIK-2 were expressed in the proximal tubule (PT), thick ascending limb (TAL), connecting tubule (CNT), and cortical collecting duct (CCD). In human kidney THIK-1 and THIK-2 were localized in PT, TAL and CCD. Immunostaining of rat tissue revealed an intracellular expression of THIK-1 and THIK-2 throughout the identified nephron segments. However in mouse kidney THIK-2 was identified in basolateral membranes. Overall, the glomerulus, thin limbs and medullary collecting ducts were devoid of THIK-1 and THIK-2 signal. In summary, THIK-1 and THIK-2 are abundantly expressed in the proximal and distal nephron of the mammalian kidney.     

Hemodynamics influences vascular peroxynitrite formation: Implication for low-density lipoprotein apo-B-100 nitration

Hemodynamics, specifically, fluid shear stress, modulates the focal nature of atherogenesis. Superoxide anion (O2(-.)) reacts with nitric oxide (.NO) at a rapid diffusion-limited rate to form peroxynitrite (O2(-.) + .NO-->ONOO(-)). Immunohistostaining of human coronary arterial bifurcations or curvatures, where OSS develops, revealed the presence of nitrotyrosine staining, a fingerprint of peroxynitrite; whereas in straight segments, where PSS occurs, nitrotyrosine was absent. We examined vascular nitrative stress in models of oscillatory (OSS) and pulsatile shear stress (PSS). Bovine aortic endothelial cells (BAEC) were exposed to fluid shear stress that simulates arterial blood flow: (1) PSS at a mean shear stress (tau(ave)) of 23 dyn cm(-2) and a temporal gradient (partial differential(tau)/partial differential(t)) at 71 dyn cm(-2) s(-1), and (2) OSS at tau(ave) = 0.02 dyn cm(- 2) and partial differential(tau)/partial differential(t) = +/- 3.0 dyn cm(-2) s(-1) at a frequency of 1 Hz. OSS significantly up-regulated one of the NADPH oxidase subunits (NOx4) expression accompanied with an increase in O2(-.) production. In contrast, PSS up-regulated eNOS expression accompanied with .NO production (total NO(2)(-) and NO(3)(-)). To demonstrate that O2(-.) and .NO are implicated in ONOO(-) formation, we added low-density lipoprotein cholesterol (LDL) to the medium in which BAEC were exposed to the above flow conditions. The medium was analyzed for LDL apo-B-100 nitrotyrosine by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS). OSS induced higher levels of 3-nitrotyrosine, dityrosine, and o-hydroxyphenylalanine compared with PSS. In the presence of ONOO(-), specific apo-B-100 tyrosine residues underwent nitration in the alpha and beta helices: alpha-1 (Tyr(144)), alpha-2 (Tyr(2524)), beta-2 (Tyr(3295)), alpha-3 (Tyr(4116)), and beta-2 (Tyr(4211)). Hence, the characteristics of shear stress in the arterial bifurcations influenced the relative production of O2(-.) and .NO with an implication for ONOO(-) formation as evidenced by LDL protein nitration.     

alpha(2)-adrenergic receptor-mediated increase in NO production buffers renal medullary vasoconstriction

The present study was designed to investigate the role of nitric oxide (NO) in modulating the adrenergic vasoconstrictor response of the renal medullary circulation. In anesthetized rats, intravenous infusion of norepinephrine (NE) at a subpressor dose of 0.1 microgram. kg(-1). min(-1) did not alter renal cortical (CBF) and medullary (MBF) blood flows measured by laser-Doppler flowmetry nor medullary tissue PO(2) (P(m)O(2)) as measured by a polarographic microelectrode. In the presence of the NO synthase inhibitor nitro-L-arginine methyl ester (L-NAME) in the renal medulla, intravenous infusion of NE significantly reduced MBF by 30% and P(m)O(2) by 37%. With the use of an in vivo microdialysis-oxyhemoglobin NO-trapping technique, we found that intravenous infusion of NE increased interstitial NO concentrations by 43% in the renal medulla. NE-stimulated elevations of tissue NO were completely blocked either by renal medullary interstitial infusion of L-NAME or the alpha(2)-antagonist rauwolscine (30 microgram. kg(-1). min(-1)). Concurrently, intavenous infusion of NE resulted in a significant reduction of MBF in the presence of rauwolscine. The alpha(1)-antagonist prazosin (10 microgram. kg(-1). min(-1) renal medullary interstitial infusion) did not reduce the NE-induced increase in NO production, and NE increased MBF in the presence of prazosin. Microdissection and RT-PCR analyses demonstrated that the vasa recta expressed the mRNA of alpha(2B)-adrenergic receptors and that medullary thick ascending limb and collecting duct expressed the mRNA of both alpha(2A)- and alpha(2B)-adrenergic receptors. These subtypes of alpha(2)-adrenergic receptors may mediate NE-induced NO production in the renal medulla. We conclude that the increase in medullary NO production associated with the activation of alpha(2)-adrenergic receptors counteracts the vasoconstrictor effects of NE in the renal medulla and may play an important role in maintaining a constancy of MBF and medullary oxygenation.     

Polyol determination along the rat nephron

The polyols sorbitol and inositol were determined in single freshly microdissected tubule segments of rat kidney. Twenty different structures were separated from six different kidney zones reaching from cortex to papillary tip. Picomol amounts of sorbitol and inositol were quantitated by use of an enzymatic bioluminescence procedure. Experimental conditions (700 mosmol/kg, 4 degrees C) were chosen to assure constant polyol concentrations over 3 h dissection period. Sorbitol exhibited a concentration gradient in the collecting duct system from the outer/inner medullary border (3.9 +/- 0.5 pmol/mm) to the papillary tip (78.8 +/- 6.9 pmol/mm). In the same region descending and ascending limbs of Henle's loop contained 1.5 +/- 0.5 to 5.3 +/- 1.6 pmol/mm and 2.5 +/- 0.8 to 8.35 +/- 1.5 pmol/mm, respectively. In contrast, all outer medullary and cortical structures had lower sorbitol concentrations. Inositol amounts increased continuously in the collecting duct from cortex (5.3 +/- 0.5 pmol/mm) to inner medulla (30.7 +/- 3.8 pmol/mm). This polyol was also found in thick ascending limb of Henle's loop (6.2 +/- 1.1 pmol/mm in cortex to 11.2 +/- 1.4 pmol/mm in outer medulla) and in proximal tubules (5.6 +/- 1.2 pmol/mm in S1 and 4.5 +/- 1.5 pmol/mm in S3). When related to cellular volume measured by planimetry, intracellular sorbitol concentration was calculated to be 51 mmol/l in papillary collecting duct and inositol 28 mmol/l in outer medullary thick ascending limb cells. These data confirm the role of sorbitol in the renal concentrating process in papilla. Inositol seems to have additional function in thick ascending limb of Henle's loop and the proximal tubule.     

Vascular smooth muscle cell glycocalyx modulates shear-induced proliferation, migration, and NO production responses

The endothelial cell glycocalyx, a structure coating the luminal surface of the vascular endothelium, and its related mechanotransduction have been studied by many over the last decade. However, the role of vascular smooth muscle cells (SMCs) glycocalyx in cell mechanotransduction has triggered little attention. This study addressed the role of heparan sulfate proteoglycans (HSPGs), a major component of the glycocalyx, in the shear-induced proliferation, migration, and nitric oxide (NO) production of the rat aortic smooth muscle cells (RASMCs). A parallel plate flow chamber and a peristaltic pump were employed to expose RASMC monolayers to a physiological level of shear stress (12 dyn/cm(2)). Heparinase III (Hep.III) was applied to selectively degrade heparan sulfate on the SMC surface. Cell proliferation, migration, and NO production rates were determined and compared among the following four groups of cells: 1) untreated with no flow, 2) Hep.III treatment with no flow, 3) untreated with flow of 12 dyn/cm(2) exposure, and 4) Hep.III treatment with flow of 12 dyn/cm(2) exposure. It was observed that flow-induced shear stress significantly suppressed SMC proliferation and migration, whereas cells preferred to aligning along the direction of flow and NO production were enhanced substantially. However, those responses were not found in the cells with Hep.III treatment. Under flow condition, the heparinase III-treated cells remained randomly oriented and proliferated as if there were no flow presence. Disruption of HSPG also enhanced wound closure and inhibited shear-induced NO production significantly. This study suggests that HSPG may play a pivotal role in mechanotransduction of SMCs.     

Resveratrol Increases Nitric Oxide Production in the Rat Thick Ascending Limb via Ca2+/Calmodulin

The thick ascending limb of the loop of Henle reabsorbs 30% of the NaCl filtered through the glomerulus. Nitric oxide (NO) produced by NO synthase 3 (NOS3) inhibits NaCl absorption by this segment. Resveratrol, a polyphenol, has beneficial cardiovascular and renal effects, many of which are mediated by NO. Resveratrol increases intracellular Ca²⁺ (Ca_i) and AMP kinase (AMPK) and NAD-dependent deacetylase sirtuin1 (SIRT1) activities, all of which could activate NO production. We hypothesized that resveratrol stimulates NO production by thick ascending limbs via a Ca²⁺/calmodulin-dependent mechanism. To test this, the effect of resveratrol on NO bioavailability was measured in thick ascending limb suspensions. Ca_i was measured in single perfused thick ascending limbs. SIRT1 activity and expression were measured in thick ascending limb lysates. Resveratrol (100 µM) increased NO bioavailability in thick ascending limb suspensions by 1.3±0.2 AFU/mg/min (p<0.03). The NOS inhibitor L-NAME blunted resveratrol-stimulated NO bioavailability by 96±11% (p<0.03). The superoxide scavenger tempol had no effect. Resveratrol elevated Ca_i from 48±7 to 135±24 nM (p<0.01) in single tubules. In Ca²⁺-free media, the resveratrol-induced increase in NO was blunted by 60±20% (p<0.05) and the rise in Ca_i reduced by 80%. Calmodulin inhibition prevented the resveratrol-induced increase in NO (p<0.002). AMPK inhibition had no effect. Resveratrol did not increase SIRT1 activity. We conclude that resveratrol increases NO production in thick ascending limbs via a Ca²⁺/calmodulin dependent mechanism, and SIRT1 and AMPK do not participate. Resveratrol-stimulated NO production in thick ascending limbs may account for part of its beneficial effects.     

Localization by immunofluorescence and by light- and electron-microscopic immunoperoxidase techniques of Tamm–Horsfall glycoprotein in adult hamster kidney

1. Tamm-Horsfall glycoprotein was isolated from hamster urine and antiserum against it was produced in rabbits. Immunoglobulin G was isolated from the antiserum. 2. Indirect methods of immunofluorescence staining were applied to kidney sections previously fixed by both perfusion and immersion methods. Tamm-Horsfall glycoprotein was identified associated with only the cells of the ascending limb of the loop of Henle and the distal convoluted tubule. Maculae densae were free of the glycoprotein. 3. Indirect immunoperoxidase procedures with light microscopy were applied to kidney sections. The results extended those found by immunofluorescence by showing that the glycoprotein is largely associated with the plasma membrane of the cells. Macula densa cells were shown to be free of the glycoprotein, although the luminal surface of the remaining cells in the transverse section of the nephron at that region was shown to contain it. 4. A variety of immuno-electron-microscopic techniques were applied to sections previously fixed in a number of ways. Providing periodate/lysine/paraformaldehyde was used as the fixative, the glycoprotein was often seen to be present not only on the luminal surface of the cells of the thick ascending limb of the loop of Henle and of the distal convoluted tubule, but also on the basal plasma membrane, including the infoldings. 5. It is generally accepted that the hyperosmolarity in the medulla of the kidney results from passage of Cl(-) ions with their accompanying Na(+) ions across the single cell layer of the lumen of the thick ascending limb of the loop of Henle, a region of the nephron with relatively high impermeability to water. We suggest that Tamm-Horsfall glycoprotein operates as a barrier to decrease the passage of water molecules by trapping the latter at the membrane of the cells. Our hypothesis requires the glycoprotein on the basal plasma membrane also.     

Renal adrenergic receptors: localization of [125I]prazosin binding sites along the microdissected rat nephron.

Norepinephrine stimulates renal tubular sodium reabsorption, probably through an alpha 1-adrenoceptor-mediated mechanism. Although the distribution of alpha 1-adrenoceptors in the kidney has been studied with autoradiography, the precise location of these receptors in isolated nephron segments is unclear. Using a microassay we determined the specific binding of [125I]iodoarylazidoprazosin ([125I]prazosin), a high specific radioactivity analog of the selective alpha 1-antagonist prazosin, to microdissected glomeruli and tubule segments. Specific binding of [125I]prazosin (3 nM) in the proximal convoluted tubule was time- and concentration-dependent, saturable, and reversible. In this segment the apparent KD by association and dissociation rate constants of [125I]prazosin binding was 0.47 nM, and the maximum receptor density was approximately 0.19 fmol/mm, or 720 fmol/mg protein. Binding specificity was verified in competition studies with excess (3 microM) unlabeled prazosin and probes for alpha 2- (yohimbine), beta- (propranolol), dopamine1- (SCH23390), and dopamine2- (S-sulpiride) receptors. [125I]Prazosin binding was inhibited significantly only by unlabeled prazosin. Mapping of prazosin binding along the nephron revealed that the highest density was in the proximal convoluted tubule, followed by the proximal straight tubule. Lesser binding was found in the thick ascending limb and in the distal convoluted tubule, whereas in the cortical and outer medullary collecting duct and in glomeruli, binding was not significantly different from zero.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histochemical localization of hormone sensitive adenylate cyclase in defined nephron epithelia in culture

Distal nephron epithelia of defined anatomical origin were microdissected from rabbit kidneys and individually explanted into an in vitro culture system. The 7 day monolayers grown from four different nephron epithelia were studied for the presence and amount of adenylate cyclase reaction product. In each case basal adenylate cyclase was compared with the enzyme reaction product after stimulation by arginine vasopressin, calcitonin, parathyroid hormone (PTH) and isoproterenol. In cortical collecting tubule cultures, the reaction was stimulated by vasopressin greater than isoproterenol greater than calcitonin. PTH had no effect. In cortical thick ascending loop of Henle cells, the stimulation was by calcitonin greater than vasopressin = PTH. Isoproterenol had no effect. In medullary ascending loop epithelia, stimulation was by vasopressin = calcitonin. Neither isoproterenol nor PTH had an effect. These observations indicate that adenylate cyclase is histochemically demonstrable in cultivated cells from rabbit distal nephron segments and that the enzyme activation by hormones is differential according to the epithelium of origin.