Western diets are typically high in sodium, widely regarded to increase blood pressure (BP). They are also characterised by a relatively low potassium (K) intake, also linked to higher BP. Previous studies have shown that the ability of low dietary K to increase BP is almost fully accounted for by an increase in the abundance and activity of the thiazide-sensitive sodium-chloride transporter (NCC) in the kidney distal convoluted tubule (DCT). However, there is a gap in knowledge for how NCC is increased, but also in the mechanisms underlying an accompanying hypertrophy of the DCT. This study aims to increase our understanding of the specific effects of low K intake on the molecular landscape of the DCT.

Methods: Due to the relatively low abundance of DCT cells vs. whole kidney, many DCT-specific changes are undetectable when analysing whole kidney or cortex samples. To uncover these mechanisms, Parvalbumin-GFP+ mice (GFP expressed only in DCT) were fed control (1% K) or a low K (0.2% K, n=7) diet for 4 days. Kidneys were collected on day 5, a single cell suspension prepared and GFP^– and GFP⁺ (DCT) cells separated using FACS. Cells were prepared for both bulk RNAseq and protein mass spectrometry. In parallel, kidney samples were embedded in paraffin, tubules were isolated using a laser microdissection system (LMD) and a protocol optimised to analyse these samples with mass spectrometry.

Results: Compared to control diets, mice on 0.2% K intake had increased NCC (+44%) and phosphorylated (active) NCC (+51%), and decreased expression of α-ENaC (-20%) and cleaved γ-ENaC (-10%). Mice on 0.2% K also had higher BP by tail cuff plethysmography (SBP 121.4 ± 2.1 vs. 110.8 ± 1.1 mmHg), reduced urine volume and urinary K excretion (5.5-fold). Plasma K was slightly decreased compared to control diets (4.06 vs. 4.35mmol/l). FACS of living GFP⁺ cells was optimised to 95 ± 2% purity and enrichment of DCT cells confirmed at the protein and RNA level for various DCT specific genes of interest. Mass spectrometry returned 1426 identified proteins and 75 proteins with TTest<0.05 (Control vs. LK). LK diet increases NCC as expected. Kir5.1 and Parvalbumin also increase, while V-ATPase subunits C and E decrease. Other potentially interesting proteins are involved in zinc transport, glycolysis and tubular structure. More generally, results suggest a remodelling of the DCT with DCT1 expanding and DCT2/CNT shrinking.

LMD sample analysis is ongoing. LMD studies are addressing the specific proteomes of proximal tubule cells and collecting duct cells subsequent to 0.2% K intake. 

Perspectives: Understanding the mechanisms of K handling and their effects on BP may offer new targets for prevention or treatment of hypertension, both through drug and dietary intervention. We have developed a multiplatform approach to study DCT cells by proteomics, transcriptomics and coupled it to functional studies. We have also developed a protocol for processing laser microdissection samples that allows for a good recovery of proteins from a low number of tubules.