An in vivo protein landscape of the mouse DCT during high dietary K⁺ or low dietary Na⁺ intake

Abstract

The hormone aldosterone is essential for maintaining K+ and Na+ balance and controlling blood pressure. Aldosterone has different effects if it is secreted due to hypovolemia or hyperkalemia. The kidney distal convoluted tubule (DCT) is believed to play a central role in mediating the differential responses to aldosterone. To determine the alterations in the DCT that may be responsible for these effects, male mice with GFP expression specifically in the DCT were maintained on diets containing low NaCl (hypovolemic state) or high K citrate (hyperkalemic state) for 4 days and DCT cells isolated using fluorescence-activated cell sorting. This pure population of DCT cells were subjected to analysis by liquid chromatography-coupled tandem mass spectrometry. Over 3000 proteins were identified in the DCT, creating the first proteome of the mouse DCT. Of the identified proteins, 210 were altered in abundance following a low NaCl diet and 625 following the high K+ diet. Many of these changes were not detectable by analyzing whole kidney samples from the same animals. When comparing responses to high K+ versus low Na+ diets, protein translation, chaperone-mediated protein folding and protein ubiquitylation are likely to be significantly altered in the DCT subsequent to a high K+ diet. In conclusion, this study defines an in vivo protein landscape of the DCT in male mice following either a low NaCl or a high K+ diet and acts as an essential resource for the kidney research community.

High dietary potassium causes ubiquitin-dependent degradation of the kidney sodium-chloride cotransporter

“…The balance between ubiquitin-dependent degradation and protein folding is maintained by the chaperone proteins heat shock protein 70 (Hsp70) and Hsp90 (21). In our recent largescale proteomics study, the abundance of these and other heat shock proteins was increased specifically in the mouse DCT after a high dietary K + intake (22), suggesting they play an important role in modulating NCC levels following a high K + diet. The role of Hsp70 and Hsp90 depends on their interaction with other proteins including the C-terminus of Hsc70 interacting protein (CHIP, encoded by Stub1) and the Hsp70-Hsp90 organizing protein HOP (Stip1).…”

“…Theoretically this interaction could dephosphorylate Hsp70/Hsc70 leading to association of NCC with CHIP and subsequent targeting for degradation. PP1 activity has been linked to short-term modulation of NCC function via the protein phosphatase 1 inhibitor-1 (I-1) (28,29), and the abundance of the alpha isoform of the PP1 catalytic subunit (PP1α) increases specifically in the DCT of mice fed a high K + diet for 4 days (22). However, a role of PP1 for modulating NCC abundance has not been investigated.…”

Potassium effects on NCC are attenuated during inhibition of Cullin E3-ubiquitin ligases

“…Using mass-spectrometry, Cul1 and Cul2 expression were increased in the DCT of mice fed a high K + diet for 4 days [24]. To further examine alterations in Cullin expression during high dietary K + intake, mice were fed either a 0%, 1% or 5% K + diet for 2-3 weeks (see Methods ) and the abundance of Cullins in whole kidney homogenates determined using Western blotting.…”

“…Altered plasma K + levels per se observed during altered K + intake also modulate NCC abundance and phosphorylation by modulation of the WNK-SPAK/OSR1 pathway [7]. To study the contribution of cullins in mediating K + effects on NCC independent of other systemic factors, we used our extensively characterized ex vivo renal tubule preparations [24, 42, 43] and incubated them in different concentrations of K + (2.5, 3.5 or 6 mM) for 30 min or 24 h (Fig. 4).…”

Potassium Effects on NCC Are Attenuated during Inhibition of Cullin E3–Ubiquitin Ligases

“…Using mass spectrometry, we recently discovered that Cul1 is enriched in the DCT, and Cul1 and Cul2 abundances are increased in the DCT of mice fed a high K + diet for 4 days [ 24 ]. This suggests that cullins other than Cul3 may also play a role in mediating the inhibitory effects of K + on NCC phosphorylation and abundance.…”