Depolarization of the macula densa induces superoxide production via NAD(P)H oxidase

2007 ◽  
Vol 292 (6) ◽  
pp. F1867-F1872 ◽  
Author(s):  
Ruisheng Liu ◽  
Jeffrey L. Garvin ◽  
YiLin Ren ◽  
Patrick J. Pagano ◽  
Oscar A. Carretero
Keyword(s):  
Nitric Oxide ◽  
Superoxide Dismutase ◽  
Oxidase Inhibitor ◽  
Fluorescent Dye ◽  
Macula Densa ◽  
Superoxide Production ◽  
Tubuloglomerular Feedback ◽  
Thick Ascending Limb ◽  
Loop Of Henle ◽  
Nacl Concentration

Superoxide (O2−) enhances tubuloglomerular feedback by scavenging nitric oxide at the macula densa. However, the singling pathway of O2− production in the macula densa is not known. We hypothesized that the increase in tubular NaCl concentration that initiates tubuloglomerular feedback induces O2− production by the macula densa via NAD(P)H oxidase, which is activated by macula densa depolarization. We isolated and microperfused the thick ascending limb of the loop of Henle and attached macula densa in rabbits. A fluorescent dye, dihydroethidium, was used to detect O2− production at the macula densa. When luminal NaCl was switched from 10 to 80 mM, a situation of initiating maximum tubuloglomerular feedback response, O2− production significantly increased. To make sure that the shifts in the oxyethidium/dihydroethidium ratio were due to changes in O2−, we used tempol (10−4 M), a stable membrane-permeant superoxide dismutase mimetic. With tempol present, when we switched from 10 to 80 mM NaCl, the increase in oxyethidium/dihydroethidium ratio was blocked. To determine the source of O2−, we used the NAD(P)H oxidase inhibitor apocynin. When luminal NaCl was switched from 10 to 80 mM in the presence of apocynin, O2− production was inhibited by 80%. To see whether the effect of increasing luminal NaCl involves Na-K-2Cl cotransporters, we inhibited them with furosemide. When luminal NaCl was switched from 10 to 80 mM in the presence of furosemide, O2− production was blocked. To test whether depolarization of the macula densa induces O2− production, we artificially induced depolarization by adding valinomycin (10−6 M) and 25 mM KCl to the luminal perfusate. Depolarization alone significantly increases O2− production. We conclude that increasing luminal NaCl induces O2− production during tubuloglomerular feedback. O2− generated by the macula densa is primarily derived from NAD(P)H oxidase and is induced by depolarization.

scholarly journals Intracellular pH regulates superoxide production by the macula densa

2008 ◽  
Vol 295 (3) ◽  
pp. F851-F856 ◽  
Author(s):  
Ruisheng Liu ◽  
Oscar A. Carretero ◽  
Yilin Ren ◽  
Hong Wang ◽  
Jeffrey L. Garvin
Keyword(s):  
Intracellular Ph ◽  
Physiological Saline ◽  
Oxidase Inhibitor ◽  
Macula Densa ◽  
Extracellular Ph ◽  
Superoxide Production ◽  
Tubuloglomerular Feedback ◽  
Effect Of Ph ◽  
Optimum Ph ◽  
Dimethyl Amiloride

We hypothesized that elevated macula densa intracellular pH (pHi) during tubuloglomerular feedback enhances O2−production from NAD(P)H oxidase. Microdissected thick ascending limbs from rabbits with intact macula densa were cannulated and perfused with physiological saline. When luminal NaCl was switched from 10 to 80 mM, O2−production increased from 0.53 ± 0.09 to 2.62 ± 0.54 U/min ( P < 0.01). To determine whether inhibiting the Na/H exchanger blocks O2−production, we used dimethyl amiloride (DMA) to block Na/H exchange. In the presence of DMA, O2−production induced by NaCl was blunted by 40%. To study the effect of pHion O2−in intact macula densa cells, we measured O2−while pHiwas changed by adjusting luminal pH. When the macula densa was perfused with 80 mM NaCl and the pH of the perfusate was switched to 6.8, 7.4, and 8.0, O2−production was significantly enhanced, but not at 10 mM NaCl. To ascertain the source of O2−, we used the NAD(P)H oxidase inhibitor apocynin. In the presence of apocynin (10−5M), O2−production induced by elevating pHiwas blocked. Finally, we measured the optimum pH for O2−production by the macula densa and found optimum extracellular pH is at 7.7 and optimum pHiis ∼8 for O2−production. We found that elevated pHienhances O2−production from NAD(P)H oxidase induced by increasing luminal NaCl when the lumen is perfused with 80 mM NaCl, not 10 mM, and O2−production is pH sensitive, with an optimum pHiof 8.

scholarly journals Enhanced amiloride-sensitive superoxide production in renal medullary thick ascending limb of Dahl salt-sensitive rats

2008 ◽  
Vol 295 (3) ◽  
pp. F726-F733 ◽  
Author(s):  
Paul M. O'Connor ◽  
Limin Lu ◽  
Carlos Schreck ◽  
Allen W. Cowley
Keyword(s):  
Left Kidney ◽  
Fluorescent Dye ◽  
Superoxide Production ◽  
Thick Ascending Limb ◽  
Control Strain ◽  
Production Studies ◽  
Medullary Thick Ascending Limb ◽  
Nacl Concentration ◽  
Cellular Pathways ◽  
Microscope Stage

The aims of the present study were to determine whether superoxide (O2−) production is enhanced in medullary thick ascending limb (mTAL) of Dahl salt-sensitive (SS) rats compared with a salt-resistant consomic control strain (SS.13BN) and to elucidate the cellular pathways responsible for augmented O2− production. Studies were carried out in 7- to 10-wk-old male SS and SS.13BN rats fed either a 0.4% NaCl diet or a 4.0% NaCl diet for 3 days before tissue harvest. Tissue strips containing mTAL were isolated from the left kidney, loaded with the O2−-sensitive fluorescent dye dihydroethidium, superfused with modified Hanks’ solution, and imaged at ×60 magnification on a heated microscope stage. O2− production was stimulated in mTAL by incrementing superfusate NaCl concentration from 154 to 254 to 500 mM. O2− production was enhanced in mTAL of SS rats compared with SS.13BN rats in response to incrementing bath NaCl. Addition of N-methyl-amiloride (100 μM) or inhibition of NAD(P)H oxidase reduced O2− production in SS mTAL to levels observed in SS.13BN rats. Both amiloride- and ouabain-sensitive pathways of O2− production were elevated following 3 days of high (4.0%) NaCl feeding in mTAL of SS and SS.13BN rats. We conclude that mTAL from SS rats exhibit enhanced amiloride-sensitive O2− production. The amiloride-sensitive O2− response in mTAL is independent of active Na+ transport and appears to be mediated by NAD(P)H oxidase. Amiloride-sensitive O2− production is likely to contribute to augmented outer medullary O2− production observed in SS rats during both normal and high NaCl diets.

scholarly journals Signal transduction in a compliant thick ascending limb

2012 ◽  
Vol 302 (9) ◽  
pp. F1188-F1202 ◽  
Author(s):  
Anita T. Layton ◽  
Leon C. Moore ◽  
Harold E. Layton
Keyword(s):  
Signal Transduction ◽  
Flow Speed ◽  
Macula Densa ◽  
Tubuloglomerular Feedback ◽  
Low Flow ◽  
Time Interval ◽  
Thick Ascending Limb ◽  
Variable Degree ◽  
Nacl Concentration

In several previous studies, we used a mathematical model of the thick ascending limb (TAL) to investigate nonlinearities in the tubuloglomerular feedback (TGF) loop. That model, which represents the TAL as a rigid tube, predicts that TGF signal transduction by the TAL is a generator of nonlinearities: if a sinusoidal oscillation is added to constant intratubular fluid flow, the time interval required for an element of tubular fluid to traverse the TAL, as a function of time, is oscillatory and periodic but not sinusoidal. As a consequence, NaCl concentration in tubular fluid alongside the macula densa will be nonsinusoidal and thus contain harmonics of the original sinusoidal frequency. We hypothesized that the complexity found in power spectra based on in vivo time series of key TGF variables arises in part from those harmonics and that nonlinearities in TGF-mediated oscillations may result in increased NaCl delivery to the distal nephron. To investigate the possibility that a more realistic model of the TAL would damp the harmonics, we have conducted new studies in a model TAL that has compliant walls and thus a tubular radius that depends on transmural pressure. These studies predict that compliant TAL walls do not damp, but instead intensify, the harmonics. In addition, our results predict that mean TAL flow strongly influences the shape of the NaCl concentration waveform at the macula densa. This is a consequence of the inverse relationship between flow speed and transit time, which produces asymmetry between up- and downslopes of the oscillation, and the nonlinearity of TAL NaCl absorption at low flow rates, which broadens the trough of the oscillation relative to the peak. The dependence of waveform shape on mean TAL flow may be the source of the variable degree of distortion, relative to a sine wave, seen in experimental recordings of TGF-mediated oscillations.

Nonlinear filter properties of the thick ascending limb

1997 ◽  
Vol 273 (4) ◽  
pp. F625-F634 ◽  
Author(s):  
H. E. Layton ◽  
E. Bruce Pitman ◽  
Leon C. Moore
Keyword(s):  
Steady State ◽  
Frequency Response ◽  
Transit Time ◽  
Concentration Profile ◽  
Tubuloglomerular Feedback ◽  
Thick Ascending Limb ◽  
Transport Parameters ◽  
Nodal Structure ◽  
Nacl Concentration ◽  
Fluid Transit Time

A mathematical model was used to investigate the filter properties of the thick ascending limb (TAL), that is, the response of TAL luminal NaCl concentration to oscillations in tubular fluid flow. For the special case of no transtubular NaCl backleak and for spatially homogeneous transport parameters, the model predicts that NaCl concentration in intratubular fluid at each location along the TAL depends only on the fluid transit time up the TAL to that location. This exact mathematical result has four important consequences: 1) when a sinusoidal component is added to steady-state TAL flow, the NaCl concentration at the macula densa (MD) undergoes oscillations that are bounded by a range interval envelope with magnitude that decreases as a function of oscillatory frequency; 2) the frequency response within the range envelope exhibits nodes at those frequencies where the oscillatory flow has a transit time to the MD that equals the steady-state fluid transit time (this nodal structure arises from the establishment of standing waves in luminal concentration, relative to the steady-state concentration profile, along the length of the TAL); 3) for any dynamically changing but positive TAL flow rate, the luminal TAL NaCl concentration profile along the TAL decreases monotonically as a function of TAL length; and 4) sinusoidal oscillations in TAL flow, except at nodal frequencies, result in nonsinusoidal oscillations in NaCl concentration at the MD. Numerical calculations that include NaCl backleak exhibit solutions with these same four properties. For parameters in the physiological range, the first few nodes in the frequency response curve are separated by antinodes of significant amplitude, and the nodes arise at frequencies well below the frequency of respiration in rat. Therefore, the nodal structure and nonsinusoidal oscillations should be detectable in experiments, and they may influence the dynamic behavior of the tubuloglomerular feedback system.

scholarly journals Potential role of luminal potassium in tubuloglomerular feedback.

1997 ◽  
Vol 8 (12) ◽  
pp. 1831-1837 ◽  
Author(s):  
V Vallon ◽  
H Osswald ◽  
R C Blantz ◽  
S Thomson
Keyword(s):  
Potential Role ◽  
Macula Densa ◽  
Tubuloglomerular Feedback ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Retrograde Perfusion ◽  
Luminal Membrane ◽  
K Concentration ◽  
Tubular Flow

Transport through the Na+-2Cl(-)-K+ cotransporter in the luminal membrane of macula densa cells is considered critical for tubuloglomerular feedback (TGF). Although various studies could support the importance of luminal Na+ and Cl-, the role of luminal K+ in TGF has not been thoroughly addressed. The study presented here examines this issue in nephrons with superficial glomeruli of anesthetized male Munich-Wistar-Frömter rats. Ambient Na+ concentration in early distal tubular fluid was approximately 22 mM, suggesting collection sites relatively close to the macula densa segment. First, it was found that ambient early distal tubular K+ concentration is approximately 1.3 mM, i.e., close to the K+ affinity of the Na+-2Cl(-)-K+ cotransporter in the thick ascending limb. Second, it was observed that a change in late proximal tubular flow rate, i.e., a maneuver that is known to induce a TGF response, significantly alters early distal tubular K+ concentration. Third, previous experiments failed to show an inhibition in TGF response during retrograde perfusion of the macula densa with K+-free solutions. Because of a potential K+ influx into the lumen between the perfusion site and the macula densa, however, the K+ channel blocker U37883A was added to the K+-free perfusate. TGF response was assessed as the fall in nephron filtration rate in response to retrograde perfusion of the macula densa segment from early distal tubular site. It was observed that luminal U37883A (100 microM) significantly attenuated TGF. Because adding 5 mM KCl to the perfusate restored TGF in the presence of U37883A and because the inhibitory action of U37883A on tubular K+ secretion was confirmed, the effect of U37883A on TGF was most likely caused by inhibition of K+ influx into the perfused segment, which decreased luminal K+ concentration at the macula densa. The present findings support a potential role for luminal K+ in TGF, which is in accordance with a transmission of the TGF signal across the macula densa via Na+-2Cl(-)-K+ cotransporter.

Tubuloglomerular feedback in ACE-deficient mice

1999 ◽  
Vol 276 (5) ◽  
pp. F751-F757 ◽  
Author(s):  
Timothy Traynor ◽  
Tianxin Yang ◽  
Yuning G. Huang ◽  
John H. Krege ◽  
Josie P. Briggs ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Blood Pressure Response ◽  
Signal Transmission ◽  
Macula Densa ◽  
Mutant Mice ◽  
Tubuloglomerular Feedback ◽  
Ang Ii ◽  
Null Mutant ◽  
Nacl Concentration ◽  
Null Mutant Mice

In these experiments, we used a strain of angiotensin converting enzyme (ACE) germline null mutant mice, generated by J. H. Krege and co-workers (J. H. Krege, S. W. M. John, L. L. Langenbach, J. B. Hodgin, J. R. Hagaman, E. S. Bachman, J. C. Jennette, D. A. O’Brien, and O. Smithies. Nature 375: 146–148, 1995), to examine the effect of chronic ACE deficiency on the magnitude of tubuloglomerular feedback (TGF) responses. The genotype was determined by PCR on DNA extracted from the tail and was verified after each experiment by assessment of the blood pressure response to an injection of ANG I. To assess TGF responsiveness, we determined the change in stop-flow pressure (PSF) caused by increasing NaCl concentration at the macula densa by using micropuncture techniques. When loop of Henle flow rate was increased from 0 to 40 nl/min, PSF fell from a mean of 42.3 ± 1.95 to 33.6 ± 2.09 mmHg ( n = 6, P = 0.005) in wild-type mice (+/+), fell from 40.6 ± 2.35 to 38.6 ± 1.93 mmHg in heterozygous (+/−) mice ( n = 7, P = 0.014), and did not change in homozygous ACE (−/−) mice [36.7 ± 2.02 mmHg vs. 36.4 ± 2.01 mmHg; n = 4, P = not significant (NS)]. During an infusion of ANG II at a dose that did not significantly elevate blood pressure (70 ng ⋅ kg−1 ⋅ min−1), TGF response magnitude (PSF 0 − PSF 40) increased from 6.5 ± 1.4 to 9.8 ± 1.19 mmHg in +/+ ( P = 0.006), from 1.14 ± 0.42 to 4.6 ± 1.3 mmHg in +/− ( P = 0.016), and from 0.42 ± 0.25 to 4.02 ± 1.06 in −/− mice ( P = 0.05). Absence of TGF responses in ACE null mutant mice and restoration of near-normal responses during an acute infusion of ANG II supports previous conclusions that ANG II is an essential component in the signal transmission pathway that links the macula densa with the glomerular vascular pole.

Effects of barium ions on tubuloglomerular feedback

1995 ◽  
Vol 268 (5) ◽  
pp. F960-F966 ◽  
Author(s):  
J. Schnermann
Keyword(s):  
Tubuloglomerular Feedback ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Experimental Conditions ◽  
Nacl Transport ◽  
Retrograde Perfusion ◽  
Low Concentrations ◽  
Vasoconstrictor Response ◽  
Nacl Concentration ◽  
Flow Pressure

The furosemide sensitivity of the tubuloglomerular feedback (TGF) response has suggested an important role for the Na-2Cl-K cotransporter in the mechanism by which increased luminal NaCl concentration causes afferent arteriolar vasoconstriction. The present experiments in anesthetized rats were performed to evaluate the effect of K channel blockade with Ba on TGF, since Ba has been shown to inhibit NaCl transport in the thick ascending limb. The presence of either 1.5 or 2 mM BaCl2 during retrograde perfusion with a 135 mM NaCl solution reduced the decrease of early proximal flow rate (VEP) by 2.7 +/- 0.76 (P < 0.02) and 4.2 +/- 0.8 nl/min (P < 0.01) compared with perfusion without BaCl2. Retrograde perfusion with 38 mM NaCl + 5 mM KCl reduced VEP by 10.4 +/- 1.3 nl/min, whereas 40 mM NaCl + 1.5 mM BaCl2 caused a reduction by only 6.1 +/- 1.4 nl/min (P < 0.001). In contrast to the inhibition caused by retrograde perfusion with low concentrations of BaCl2, increased vasoconstriction was seen during retrograde perfusion with 5 mM BaCl2 or during orthograde perfusion with 10 mM BaCl2. The addition of 10(-4) M furosemide to a solution containing 5 mM BaCl2 largely blocked the increased vasoconstrictor response. Peritubular perfusion with a solution containing 5 mM BaCl2 caused a fall in stop-flow pressure in an adjacent nephron by 10.7 +/- 1.5 mmHg (P < 0.001). These results indicate that under our experimental conditions Ba ions exert a dual effect on vascular responses to changes in luminal NaCl concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Luminal flow induces eNOS activation and translocation in the rat thick ascending limb

2004 ◽  
Vol 287 (2) ◽  
pp. F274-F280 ◽  
Author(s):  
Pablo A. Ortiz ◽  
Nancy J. Hong ◽  
Jeffrey L. Garvin
Keyword(s):  
Nitric Oxide ◽  
Apical Membrane ◽  
Renal Medulla ◽  
Cytochalasin D ◽  
No Synthase ◽  
No Production ◽  
Thick Ascending Limb ◽  
Loop Of Henle ◽  
Luminal Flow ◽  
Nos Inhibitor

Nitric oxide (NO) produced by endothelial NO synthase (eNOS) acts as an autacoid to inhibit NaCl absorption in the thick ascending limb of the loop of Henle (THAL). In the vasculature, shear stress activates eNOS. We hypothesized that increasing luminal flow activates eNOS and enhances NO production in the THAL. We measured NO production by isolated, perfused THALs using a NO-sensitive microelectrode. Increasing luminal flow from 0 to 20 nl/min increased NO production by 43.1 ± 4.1 pA/mm of tubule ( n = 10, P < 0.05), and this response was blunted (92%) by the NOS inhibitor l-ωnitro-methylarginine ( P < 0.05). We studied the effect of flow on eNOS subcellular localization. In the absence of flow, eNOS was diffusely localized throughout the cell (basolateral = 33 ± 4%; middle = 27 ± 3%; apical = 40 ± 4% of total eNOS). Increasing luminal flow induced eNOS translocation to the apical membrane, as evidenced by a 60% increase in eNOS immunoreactivity in the apical membrane (from 40 ± 4 to 65 ± 2%; n = 6; P < 0.05). Disrupting the actin cytoskeleton with cytochalasin D (10 μM) reduced flow-induced NO production by 62% (from 37.1 ± 3.4 to 14.0 ± 2.4 pA/mm tubule, n = 7, P < 0.04) and blocked flow-induced eNOS translocation. Flow also increased the amount of phosphorylated eNOS (Ser1179) at the apical membrane (from 25 ± 2 to 56 ± 2%; P < 0.05). We conclude that increasing luminal flow induces eNOS activation and translocation to the apical membrane in THALs. These are the first data showing that flow regulates eNOS in epithelial cells. This may be an important mechanism for regulation of NO levels in the renal medulla.

scholarly journals Salt-sensitive splice variant of nNOS expressed in the macula densa cells

2010 ◽  
Vol 298 (6) ◽  
pp. F1465-F1471 ◽  
Author(s):  
Deyin Lu ◽  
Yiling Fu ◽  
Arnaldo Lopez-Ruiz ◽  
Rui Zhang ◽  
Ramiro Juncos ◽  
...  
Keyword(s):  
Salt Intake ◽  
Splice Variants ◽  
Macula Densa ◽  
High Salt ◽  
Tubuloglomerular Feedback ◽  
No Production ◽  
Thick Ascending Limb ◽  
High Salt Diet ◽  
Salt Diet ◽  
High Salt Intake

Neuronal nitric oxide synthase (nNOS), which is abundantly expressed in the macula densa cells, attenuates tubuloglomerular feedback (TGF). We hypothesize that splice variants of nNOS are expressed in the macula densa, and nNOS-β is a salt-sensitive isoform that modulates TGF. Sprague-Dawley rats received a low-, normal-, or high-salt diet for 10 days and levels of the nNOS-α, nNOS-β, and nNOS-γ were measured in the macula densa cells isolated with laser capture microdissection. Three splice variants of nNOS, α-, β-, and γ-mRNAs, were detected in the macula densa cells. After 10 days of high-salt intake, nNOS-α decreased markedly, whereas nNOS-β increased two- to threefold in the macula densa measured with real-time PCR and in the renal cortex measured with Western blot. NO production in the macula densa was measured in the perfused thick ascending limb with an intact macula densa plaque with a fluorescent dye DAF-FM. When the tubular perfusate was switched from 10 to 80 mM NaCl, a maneuver to induce TGF, NO production by the macula densa was increased by 38 ± 3% in normal-salt rats and 52 ± 6% ( P < 0.05) in the high-salt group. We found 1) macula densa cells express nNOS-α, nNOS-β, and nNOS-γ, 2) a high-salt diet enhances nNOS-β, and 3) TGF-induced NO generation from macula densa is enhanced in high-salt diet possibly from nNOS-β. In conclusion, we found that the splice variants of nNOS expressed in macula densa cells were α-, β-, and γ-isoforms and propose that enhanced level of nNOS-β during high-salt intake may contribute to macula densa NO production and help attenuate TGF.

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