Journal: American Journal of Physiology - Renal Physiology

Article Title: Tamm-Horsfall protein/uromodulin deficiency elicits tubular compensatory responses leading to hypertension and hyperuricemia

doi: 10.1152/ajprenal.00233.2017

Figure Lengend Snippet: Effects of THP loss on the apical translocation of NKCC2. A: lipid raft flotation assay of the partitioning of NKCC2 in the lipid rafts. Triton X-100-insoluble protein extracts from 12-mo-old THP KO mice and age-matched WT mice were centrifuged on a stepwise sucrose density gradient (5–40% in 5% increments), and equal volumes from different fractions (1–9: lighter density fractions to the left and heavier density fractions to the right) were resolved on an SDS-PAGE and immunoblotted with anti-NKCC2 and reblotted with anti-flotillin, a lipid raft marker, and anti-THP. The results are representative of 3 similar experiments. Note the lack of partitioning of NKCC2 in the lighter fractions (6, 7) in THP KO mice, as opposed to the situation in WT mice and the coexistence of NKCC2 and THP in the same fractions in WT mice. Short bars on the left denote positions of molecular mass standards (from top to bottom): 170 and 135 kDa; 55 and 40 kDa; 135 and 100 kDa; 170 and 135-kDa; 55 and 40 kDa; 135 and 100 kDa. B, top: cellular fractionation and Western blotting. Renal cells from aged WT and THP KO mice (n = 3 per genotype) were fractionated into plasma membrane-enriched and cytosol-enriched fractions. The resultant fractions were dissolved in SDS-PAGE loading buffer and analyzed by Western blotting using anti-NKCC2 and anti-p-NKCC2 antibodies. Anti-flotillin and anti-β-actin served as loading controls for membrane and cytosol, respectively. Short bars on the right denote positions of molecular mass standards (from top to bottom): 170 and 135 kDa; 170 and 135 kDa; 55 and 40 kDa. B, bottom: densitometry of the Western blotting results shown is top, expressed as ratio to flotillin or -β−actin. Note the remarked reduction of NKCC2 and p-NKCC2 in the membrane fraction and the significant increase of NKCC2 in the cytosol. NS, no statistical significance. C: confocal immunofluorescent microcopy showing colocalization of NKCC2 with membrane marker flotillin at the apical surface of TAL of aged WT mice and, in contrast, the reduced apical labeling and increased cytoplasmic labeling of NKCC2 in the TAL of aged THP KO mice. All panels are of the same magnification; bar = 30 μm: ×400. D: immunoelectron microscopy of NKCC2 of 12-mo old THP KO mice and age-matched WT mice showing apical association of NKCC2 in WT mice and the cytoplasmic staining of NKCC2 in THP KO mice. Please refer to materials and methods for the operationally defined “Apical membrane” and “Cytoplasm”, and procedures of enumeration and tabulation of the gold particles in these two compartments. Bar = 200 nm. Bottom: average number of gold particles and SD per counted area in the apical membrane and cytoplasm.

Article Snippet: Immunoelectron microscopy/gold-colloid staining revealed that NKCC2 was closely associated with the TAL’s luminal membrane in WT mice but was largely absent in the same structure in aged THP KO mice ( , dashed ovals).

Techniques: Translocation Assay, SDS Page, Marker, Cell Fractionation, Western Blot, Clinical Proteomics, Membrane, Labeling, Immuno-Electron Microscopy, Staining