Intracellular pH regulates superoxide production by the macula densa

Area of Science:

• Renal Physiology
• Cellular Biology
• Biochemistry

Background:

• Tubuloglomerular feedback (TGF) is a key intrarenal mechanism regulating glomerular filtration rate.
• Macula densa cells play a critical role in sensing luminal NaCl concentration and initiating TGF.
• The precise molecular mechanisms linking macula densa cell signaling to TGF, including reactive oxygen species (ROS) production, are not fully elucidated.

Purpose of the Study:

• To investigate the hypothesis that elevated macula densa intracellular pH (pH(i)) enhances superoxide (O(2)(-)) production via NAD(P)H oxidase during TGF.
• To determine the role of the Na/H exchanger in regulating pH(i) and subsequent O(2)(-) production.
• To identify the optimal pH conditions for O(2)(-) production in macula densa cells.

Main Methods:

• Microdissection and cannulation of rabbit thick ascending limbs with intact macula densa.
• Perfusion with varying luminal NaCl concentrations and pH adjustments to manipulate intracellular pH.
• Measurement of O(2)(-) production using fluorescence assays, with inhibition studies using dimethyl amiloride (DMA) and apocynin.

Main Results:

• Increasing luminal NaCl from 10 to 80 mM significantly increased O(2)(-) production (0.53 to 2.62 U/min).
• Inhibition of the Na/H exchanger with DMA blunted NaCl-induced O(2)(-) production by 40%.
• Elevated luminal pH (7.4-8.0) at 80 mM NaCl enhanced O(2)(-) production, which was blocked by the NAD(P)H oxidase inhibitor apocynin, indicating pH(i) sensitivity with an optimum around pH 8.

Conclusions:

• Elevated macula densa intracellular pH enhances O(2)(-) production from NAD(P)H oxidase, particularly under conditions of high luminal NaCl.
• The Na/H exchanger plays a role in regulating macula densa pH(i) and subsequent ROS production.
• These findings highlight the pH-dependent regulation of ROS production in macula densa cells, implicating NAD(P)H oxidase in TGF signaling.