Submitted on June 17, 2008
Revised on January 21, 2009
Accepted on January 22, 2009
Low salt concentrations activate AMP-activated protein kinase in mouse macula densa cells
Natasha Cook1, Scott A Fraser2, Marina Katerelos3, Frosa Katsis4, Kurt Gleich2, Peter F Mount1, Gregory R. Steinberg5, Vicki Levidiotis6, Bruce E. Kemp5, and David A Power7*
1 Austin Health
2 Burnet Institute
3 Burnet Instuiute
4 St. Vincent's Institute of Medical Research
5 St. Vincent's Institute
6 Royal Price Alfred Hospital
7 Austin & Repatriation Medical Centre
* To whom correspondence should be addressed. E-mail: david.power{at}austin.org.au.
The energy-sensing kinase AMP-activated protein kinase (AMPK) is associated with the sodium co-transporter 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 iso-osmolar 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 iso-osmolar low salt. The specific CaMKK 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 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 iso-osmolar 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.
