SPAK Mediated NCC Regulation in Response to Low K + Diet 7 8 9

40 41 The NaCl cotransporter (NCC) of the renal distal convoluted tubule is stimulated by low 42 K + diet by an unknown mechanism. Since recent work has shown that the STE20/SPS-1-43 related proline-alanine-rich protein kinase (SPAK) can function to stimulate NCC by 44 phosphorylation of specific N-terminal sites, we investigated if the NCC response to low 45 K + diet is mediated by SPAK. Using phospho-specific antibodies in Western blot and 46 immunolocalization studies of wild-type (WT) and SPAK knockout (SPAK-/-) mice fed 47 a low K + or control diet for 4 days, we found that low K + diet strongly increased total 48 NCC expression and phosphorylation of NCC. This was associated with an increase in 49 total SPAK expression in cortical homogenates and an increase in phosphorylation of 50 SPAK at the S383 activation site. The increased pNCC in response to low K + diet was 51 blunted but not completely inhibited in SPAK-/-mice. These findings reveal that SPAK is 52 an important mediator of the increased NCC activation by phosphorylation that occurs in 53 the DCT in response to a low K + diet, but other low potassium-activated kinases are 54 likely to be involved.