NaCl Absorption In Thick Ascending Limb Research Articles

Fructose stimulates phosphorylation and trafficking of the Na/K/2Cl cotransporter in rat thick ascending limbs (1109.2)

High fructose consumption affects renal salt handling and is linked to salt‐sensitive hypertension in humans and rodents. The thick ascending limb (TAL) reabsorbs 25% of filtered NaCl via NKCC2. However, the effect of fructose on NKCC2 and TAL NaCl absorption is unknown. We hypothesized that acute and chronic fructose treatment stimulate NKCC2 activity and trafficking in TALs. Acute fructose treatment (20 min) enhanced surface NKCC2 expression (Fructose 1mM= 22 ± 5%, 5mM= 49 ± 10%, 10mM= 101 ± 59%; p<0.05), but did not increase NKCC2 phosphorylation at Ser126, Thre96/101, nor total NKCC2 expression. Fructose (5mM) increased NaCl transport‐related TAL oxygen consumption by 11 ± 3% (p<0.05), and this effect was absent when NKCC2 was blocked by furosemide. In contrast, glucose (5 mM) did not affect surface NKCC2 nor TAL oxygen consumption. When Sprague‐Dawley rats were fed 20% fructose for 1 week, surface‐to‐intracellular NKCC2 increased by 61 ± 23% (p<0.05), Thre96,101 phosphorylation increased by 8 ± 3 fold (p<0.05) but total NKCC2 expression was reduced by 40 ± 9% (p<0.05). We concluded that acute fructose treatment increases surface NKCC2 expression and NaCl transport by TALs. One week on a fructose enriched diet increased NKCC2 trafficking and phosphorylation at Thre96/101. Our data suggest that fructose but not glucose may contribute to salt‐sensitive hypertension by stimulating NKCC2 and NaCl absorption.Grant Funding Source: 1P01HL090550

Endothelin-1 Inhibits Thick Ascending Limb Transport via Akt-stimulated Nitric Oxide Production

Endothelin-1 inhibits sodium reabsorption in the thick ascending limb (THAL) via stimulation of nitric oxide (NO) production. The mechanism whereby endothelin-1 stimulates THAL NO is unknown. We hypothesized that endothelin-1 stimulates THAL NO production by activating phosphatidylinositol 3-kinase (PI3K), stimulating Akt activity, and phosphorylating NOS3 at Ser1177. This enhances NO production and inhibits sodium transport. We measured 1) NO production by fluorescence microscopy using DAF2-DA, 2) Akt activity using a fluorescence resonance energy transfer-based Akt reporter, 3) phosphorylated NOS3 and Akt by Western blotting, and 4) NKCC2 activity by fluorescence microscopy. In isolated THAL, endothelin-1 (1 nmol/liter) increased NO production from 0.23 +/- 0.24 to 2.81 +/- 0.32 fluorescence units/min (p < 0.001; n = 5) but failed to stimulate NO production in THALs isolated from NOS3-/- mice. Wortmannin (150 nmol/liter), a PI3K inhibitor, reduced endothelin-1-stimulated NO by 83% (0.49 +/- 0.13 versus 3.31 +/- 0.49 fluorescence units/min for endothelin-1 alone; p < 0.006; n = 5). Endothelin-1 stimulated Akt activity by 0.16 +/- 0.02 arbitrary units as measured by fluorescence resonance energy transfer (p < 0.001; n = 5) and increased phosphorylation of Akt at Ser473 by 56 +/- 11% (p < 0.002; n = 7). Dominant-negative Akt blocked endothelin-1-induced NO by 60 +/- 8% (p < 0.001 versus control; n = 6), and an Akt inhibitor had a similar effect. Endothelin-1 increased phosphorylation of NOS3 at Ser1177 by 89 +/- 24% (p < 0.01; n = 7) but had no effect on Ser633. Endothelin-1 inhibited NKCC2 activity, an effect that was blocked by dominant-negative Akt and NOS inhibition. We conclude that endothelin-1 stimulates THAL NO production by activating PI3K, stimulating Akt activity, and phosphorylating NOS3 at Ser1177. This enhances NO production and inhibits sodium transport.

Angiotensin II‐induced hypertension stimulates thick ascending limb NaCl absorption by increasing O2−

Renal medullary O2− increases in angiotensin‐induced hypertensive (Ang‐IH) rats. We have shown that O2− increases thick ascending limb (TAL) Na transport, but the effect of Ang‐IH on the TAL is unknown. We hypothesized that TAL Na absorption in Ang‐IH rats is enhanced due to elevated O2− levels and protein kinase C (PKC) α activity. We measured O2− levels, furosemide‐sensitive oxygen consumption (FSQO2) as a measure of Na transport and PKCα activity in TAL suspensions from control and Ang‐IH rats. In TALs from Ang‐IH rats O2− levels were 2.5±0.5 compared to 0.8±0.3 arbitrary units (AU)/min/mg protein for controls (p&lt;0.05). FSQO2 also increased (25±3 to 36±2 % of total QO2; p&lt;0.05). Tempol, a O2− scavenger, infused into 1 kidney of Ang‐IH rats decreased O2− levels from 2.3±0.4 to 1.3±0.2 AU/min/mg protein (p&lt;0.05) and FSQO2 from 33±2 to 24±2 % of total QO2 (p&lt;0.05). Tempol had no effect on O2− or FSQO2 in controls. Tempol did not alter blood pressure of Ang‐IH rats. In Ang‐IH rats PKCα activity was 3.7±0.9 compared to 1.1±0.2 AU/mg protein (p&lt;0.05) in controls. Tempol infusion into 1 kidney of Ang‐IH rats reduced PKCα activity from 3.0±0.6 to 0.6±0.1 AU/mg protein (p&lt;0.05). Infusion of the PKCα inhibitor Gö6976 into one kidney of Ang‐IH rats reduced FSQO2 from 38±3 to 26±3 % of total QO2 (p&lt;0.05). We conclude that in Ang‐IH rats TAL NaCl absorption is enhanced due to elevated O2− and PKCα activity rather than increased blood pressure.

The role of reactive oxygen species in the regulation of tubular function

The phrase reactive oxygen species covers a number of molecules and atoms, including the quintessential member of the group, O2-; singlet oxygen; H2O2; organic peroxides; and OONO-. While nitric oxide (NO) is also technically a member of the reactive oxygen species family, it is generally considered with a different class of compounds and will not be considered here. To our knowledge, there are currently no published data reporting the effects of reactive oxygen species on net transepithelial flux in the proximal nephron. However, there is evidence that OONO- regulates Na+/K+ adenosine triphosphatase (ATPase) activity as well as paracellular permeability. While it is easy to speculate that such an effect on the pump would decrease net transepithelial solute and water reabsorption, one cannot do so without knowing how other transporters are affected. O2- stimulates NaCl absorption by the thick ascending limb by activating protein kinase C and blunting the effects of NO. The effects of O2- on thick ascending limb NaCl absorption may be important for the initiation of salt-sensitive hypertension. To our knowledge, there are no published data concerning the role of reactive oxygen species in the regulation of solute absorption in either the distal convoluted tubule or the collecting duct. However, OONO- inhibits basolateral K+ channels in the cortical collecting duct, although the net effect of such inhibition is unknown. While the regulation of tubular transport by reactive oxygen species is important to overall salt and water balance, we know very little about where and how these regulators act along the nephron.