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

Abstract

The aims of the present study were to determine whether superoxide (O(2)(-)) production is enhanced in medullary thick ascending limb (mTAL) of Dahl salt-sensitive (SS) rats compared with a salt-resistant consomic control strain (SS.13(BN)) and to elucidate the cellular pathways responsible for augmented O(2)(-) production. Studies were carried out in 7- to 10-wk-old male SS and SS.13(BN) 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 O(2)(-)-sensitive fluorescent dye dihydroethidium, superfused with modified Hanks' solution, and imaged at x60 magnification on a heated microscope stage. O(2)(-) production was stimulated in mTAL by incrementing superfusate NaCl concentration from 154 to 254 to 500 mM. O(2)(-) production was enhanced in mTAL of SS rats compared with SS.13(BN) rats in response to incrementing bath NaCl. Addition of N-methyl-amiloride (100 muM) or inhibition of NAD(P)H oxidase reduced O(2)(-) production in SS mTAL to levels observed in SS.13(BN) rats. Both amiloride- and ouabain-sensitive pathways of O(2)(-) production were elevated following 3 days of high (4.0%) NaCl feeding in mTAL of SS and SS.13(BN) rats. We conclude that mTAL from SS rats exhibit enhanced amiloride-sensitive O(2)(-) production. The amiloride-sensitive O(2)(-) response in mTAL is independent of active Na(+) transport and appears to be mediated by NAD(P)H oxidase. Amiloride-sensitive O(2)(-) production is likely to contribute to augmented outer medullary O(2)(-) production observed in SS rats during both normal and high NaCl diets.

Reactive oxygen species as important determinants of medullary flow, sodium excretion, and hypertension

“…Recent studies have clarified one of these pathways, which involves H ϩ transport in the mTAL that when activated stimulates a Nox-mediated production of O 2 ·Ϫ independently of active Na ϩ transport (85,139). By utilizing SS HV1…”

“…It is the imbalance between both reduced NO and elevated O 2 ·Ϫ (and H 2 O 2 ) production in SS rats that appears to tilt the balance toward a state of oxidative stress even before a switch to a high-salt diet, which accelerates this process and leads to the rapid rise in arterial pressure and renal injury (32,130,133,139).…”

NAD(P)H oxidase and renal epithelial ion transport

“…Both NOX2 and NOX4 have been detected in mTAL, and further studies will be required to determine which of these isoforms are activated by H ϩ efflux. Importantly, O 2 Ϫ production observed in response to a number of stimuli, as well as basal ROS levels in the outer medulla, are sensitive to dimethyl amiloride, indicating that this pathway is likely an important source of ROS in vivo (22,31,32). Further, the activity of these H ϩ currents has been found to be greater in mTAL from Dahl salt-sensitive rats compared with a saltresistant control strains, indicating a potential role of this transporter toward the development of oxidative stress in the kidney and salt-sensitive hypertension (31).…”

Proton channels and renal hypertensive injury: a key piece of the Dahl salt-sensitive rat puzzle?

“…Importantly, we found that the rate of O 2 ·Ϫ production in SS mTAL was greater than that of SS. 13 BN in response to increments in extracellular NaCl concentration (59). Furthermore, this difference was enhanced in mTAL taken from rats 3 days after the advent of high-salt feeding (59).…”

“…To determine whether NADPH oxidase-dependent O 2 ·Ϫ production was elevated in medullary thick ascending limb (mTAL) tubular segments from Dahl SS, we utilized live-cell imaging of microdissected nephron segments loaded with the free radical indicator dihydroethidium (DHE) and compared the relative rates of O 2 ·Ϫ production between mTAL from SS and SS.13 BN rats (59). Given our hypothesis that "elevated O 2 ·Ϫ production resulted from increased Na ϩ delivery and tubular work following initiation of high-salt feeding," the most physiologically relevant stimuli would have been to perfuse isolated nephron segments at various flow rates to mimic alterations in Na ϩ intake and luminal NaCl delivery.…”