Gitelman's syndrome (GS) is a rare autosomal disease characterized by electrolytic alterations. Clinical features are hyperkalemia, hypomagnesemia and hypocalciuria, and hypo- or normotension notwithstanding marked activation of the renin-angiotensin-aldosterone system. These abnormalities are due to mutations affecting the thiazide sensitive Na-Cl cotransporter (NCC), which reabsorbs sodium in the distal convoluted tubule of the nephron. Over 100 mutations are known, but the molecular mechanisms underlying the impaired function remained unclear.Objective:
In our cohort of GS a young woman presented with a NCC point mutation (c.1204G > A) never reported before, which causes an aminoacid exchange (Gly394Asp). Therefore, we used a molecular biology approach to investigate how this mutation affected NCC functionality.Design and method:
We created different expression vectors containing either the wild type or the mutated NCC (SLC12A3) coding sequence. The DNA was then transfected into HEK 293 cells and RNA into Xenopus laevis oocytes. We then assessed the expression, maturation, and the trafficking of the protein by western blot, immunohistochemistry (IHC), and confocal microscopy, and the NCC functionality by Na22+ uptake.Results:
Western blots show more expressed glycosylation bands in wild type than in mutated NCC at 24 (p = 0,003 NCC wt mean + SEM 0,233 ± 0,046; NCC mut mean + SEM 0,0268 ± 0,008) and 48 hours after transfection (p = 0,0003 NCCwt 0,524 ± 0,084; NCC mut 0,059 ± 0,012), suggesting impaired maturation of the NCC mutated protein. This result was confirmed with confocal microscopy in transfected oocytes that showed apical NCC staining in wild type and NCC retention in the inner portion in mutated oocytes. Furthermore, preliminary Na22+ uptake experiments prove impaired function of the mutated NCC compared to wild type NCC.Conclusions:
These results suggest that this novel GS mutation leads impaired NCC glycosylation with ensuing retention in the endoplasmic reticulum, compromised trafficking to the plasma membrane, and diminished Na reabsorption.