Increasing NaCl Induces Superoxide Production in Macula Densa Cell Line

2007 ◽  
Vol 21 (5) ◽  
Author(s):  
Ruisheng Liu ◽  
Chunxia Lin ◽  
Oscar A. Carretero ◽  
YiLin Ren ◽  
Pamela Harding
Keyword(s):  
Cell Line ◽  
Macula Densa ◽  
Superoxide Production

scholarly journals Establishment of a Mouse Macula Densa Cell Line with an nNOS Promoter Driving EGFP Expression

2005 ◽  
Vol 55 (6) ◽  
pp. 365-372 ◽  
Author(s):  
Yukiko Yasuoka ◽  
Hideaki Kawada ◽  
Yoshiro Suzuki ◽  
Masahiro Sato ◽  
Hitoshi Endou ◽  
...  
Keyword(s):  
Cell Line ◽  
Macula Densa ◽  
Egfp Expression

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.

Renal localization and regulation of 15-hydroxyprostaglandin dehydrogenase

2008 ◽  
Vol 294 (2) ◽  
pp. F433-F439 ◽  
Author(s):  
Bing Yao ◽  
Jie Xu ◽  
Raymond C. Harris ◽  
Ming-Zhi Zhang
Keyword(s):  
Proximal Tubule ◽  
Cell Line ◽  
Collecting Duct ◽  
Macula Densa ◽  
Immunofluorescent Staining ◽  
Kidney Cortex ◽  
Proximal Tubule Cell ◽  
Thick Ascending Limb ◽  
Key Enzyme ◽  
Cox 2

Tissue prostaglandin levels are determined by both biosynthesis and catabolism. The current studies report the expression and localization of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a key enzyme in prostaglandin catabolism in the kidneys. We also investigated potential interactions between 15-PGDH and cyclooxygenase (COX), a key enzyme in prostaglandin biosynthesis. Both 15-PGDH mRNA and protein levels were significantly higher in kidney cortex than in papilla, which is opposite to the expression pattern of COX-2. In situ hybridization indicated that 15-PGDH mRNA was mainly localized to the tubular epithelial cells in kidney cortex and outer medulla but not in the glomerulus or papilla. Dual immunofluorescent staining indicated that 15-PGDH was expressed in the proximal tubule, cortical, and outer medullary thick ascending limb and collecting duct but not in the macula densa or papilla. 15-PGDH levels were significantly lower in a macula densa cell line (MMDD1) than in a proximal tubule cell line. Although a high-salt diet decreased COX-2 expression in macula densa, it increased macula densa 15-PGDH expression in both mouse and rat kidneys. In MMDD1 cells, a COX-2 inhibitor increased 15-PGDH, whereas a COX-1 inhibitor had no effect. Furthermore, intense 15-PGDH immunofluorescent staining was found in both macula densa and glomerulus in COX-2 knockout mice. The intrarenal distribution of 15-PGDH and its interactions with COX-2 suggest that differential regulation of COX-2 and 15-PGDH may play an important role in determining levels of prostaglandins involved in regulation of salt, volume, and blood pressure homeostasis.

scholarly journals Heme oxygenase attenuates angiotensin II-mediated superoxide production in cultured mouse thick ascending loop of Henle cells

2008 ◽  
Vol 295 (4) ◽  
pp. F1158-F1165 ◽  
Author(s):  
Silvia Kelsen ◽  
Bijal J. Patel ◽  
Lawson B. Parker ◽  
Trinity Vera ◽  
John M. Rimoldi ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Carbon Monoxide ◽  
Angiotensin Ii ◽  
Cell Line ◽  
Heme Oxygenase ◽  
Superoxide Production ◽  
Loop Of Henle ◽  
Ang Ii ◽  
Cobalt Protoporphyrin ◽  
Immortalized Cell

Heme oxygenase (HO)-1 induction can attenuate the development of angiotensin II (ANG II)-dependent hypertension. However, the mechanism by which HO-1 lowers blood pressure is not clear. The goal of this study was to test the hypothesis that induction of HO-1 can reduce the ANG II-mediated increase in superoxide production in cultured thick ascending loop of Henle (TALH) cells. Studies were performed on an immortalized cell line of mouse TALH (mTALH) cells. HO-1 was induced in cultured mTALH cells by treatment with cobalt protoporphyrin (CoPP, 10 μM) or hemin (50 μM) or by transfection with a plasmid containing the human HO-1 isoform. Treatment of mTALH cells with 10−9 M ANG II increased dihydroethidium (DHE) fluorescence (an index of superoxide levels) from 35.5 ± 5 to 136 ± 18 relative fluorescence units (RFU)/μm2. Induction of HO-1 via CoPP, hemin, or overexpression of the human HO-1 isoform significantly reduced ANG II-induced DHE fluorescence to 64 ± 5, 64 ± 8, and 41 ± 4 RFU/μm2, respectively. To determine which metabolite of HO-1 is responsible for reducing ANG II-mediated increases in superoxide production in mTALH cells, cells were preincubated with bilirubin or carbon monoxide (CO)-releasing molecule (CORM)-A1 (each at 100 μM) before exposure to ANG II. DHE fluorescence averaged 80 ± 7 RFU/μm2 after incubation with ANG II and was significantly decreased to 55 ± 7 and 53 ± 4 RFU/μm2 after pretreatment with bilirubin and CORM-A1. These results demonstrate that induction of HO-1 in mTALH cells reduces the levels of ANG II-mediated superoxide production through the production of both bilirubin and CO.

Low salt concentrations activate AMP-activated protein kinase in mouse macula densa cells

2009 ◽  
Vol 296 (4) ◽  
pp. F801-F809 ◽  
Author(s):  
Natasha Cook ◽  
Scott A. Fraser ◽  
Marina Katerelos ◽  
Frosa Katsis ◽  
Kurt Gleich ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cell Line ◽  
Kinase Inhibitor ◽  
Chloride Concentration ◽  
Macula Densa ◽  
Tubuloglomerular Feedback ◽  
Specific Substrate ◽  
Ampk Activation ◽  
Low Salt ◽  
Amp Activated Protein Kinase

The energy-sensing kinase AMP-activated protein kinase (AMPK) is associated with the sodium-potassium-chloride cotransporter NKCC2 in the kidney and phosphorylates it on a regulatory site in vitro. To identify a potential role for AMPK in salt sensing at the macula densa, we have used the murine macula densa cell line MMDD1. In this cell line, AMPK was rapidly activated by isosmolar low-salt conditions. In contrast to the known salt-sensing pathway in the macula densa, AMPK activation occurred in the presence of either low sodium or low chloride and was unaffected by inhibition of NKCC2 with bumetanide. Assays using recombinant AMPK demonstrated activation of an upstream kinase by isosmolar low salt. The specific calcium/calmodulin-dependent kinase kinase inhibitor STO-609 failed to suppress AMPK activation, suggesting that it was not part of the signal pathway. AMPK activation was associated with increased phosphorylation of the specific substrate acetyl-CoA carboxylase (ACC) at Ser79, as well as increased NKCC2 phosphorylation at Ser126. AMPK activation due to low salt concentrations was inhibited by an adenovirus construct encoding a kinase dead mutant of AMPK, leading to reduced ACC Ser79 and NKCC2 Ser126 phosphorylation. This work demonstrates that AMPK activation in macula densa-like cells occurs via isosmolar changes in sodium or chloride concentration, leading to phosphorylation of ACC and NKCC2. Phosphorylation of these substrates in vivo is predicted to increase intracellular chloride and so reduce the effect of salt restriction on tubuloglomerular feedback and renin secretion.

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