Nephrol Dial Transplant (2004) 19: 1069-1076
Nephrol Dial Transplant Vol. 19 No. 5 © ERA-EDTA 2004; all rights reserved
Original Article
Effects of chemical chaperones on partially retarded NaCl cotransporter mutants associated with Gitelman's syndrome in a mouse cortical collecting duct cell line
1Department of Physiology, 2Department of Biochemistry, 3Department of Pediatrics and 4Department of Human Genetics, University Medical Centre Nijmegen, Nijmegen, The Netherlands and 5INSERM U478, Institut Fédératif de Recherche 02, Faculté de Médecine Xavier Bichat, Paris, France
Correspondence and offprint requests to: René J. M. Bindels, 160 Cell Physiology, University Medical Centre Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands. Email: r.bindels{at}ncmls.kun.nl
Background. Epithelial cells lining the distal convoluted tubule express the thiazide-sensitive Na–Cl cotransporter (NCC) that is responsible for the reabsorption of 5–10% of the filtered load of Na+ and Cl–. Mutations in NCC cause the autosomal recessive renal disorder Gitelman's syndrome (GS). GS mutations give rise to mutant transporters that are either fully (class I) or partially (class II) retarded. Recent evidence indicates that class II mutations do not alter the intrinsic transport activity of NCC. These findings suggest that in GS caused by class II NCC mutations, pharmacological chaperones may be useful in treatment.
Methods. Initial attempts using 4-phenylbutyrate and glycerol to increase Na+ uptake in Xenopus laevis oocytes expressing the class II mutant L215P were unsuccessful. To study the effect of the chaperones in a more physiological setting, we next expressed hNCC in the polarized epithelial cell line of distal tubular origin, mpkCCD.
Results. mpkCCD cells readily expressed the class II mutant R955Q, but not the class I mutant G741R. Wild-type hNCC was predominantly present in the 
120–1403 kD complex glycosylated form. In contrast, the R955Q mutant was predominantly present in a lower molecular weight form of 100 kD. Pretreatment of R955Q expressing cells with 4-phenylbutyrate (5 mM, 16 h), but not thapsigargin (1 µM, 90 min), dimethyl sulfoxide (1%, 16 h) or glycerol (4%, 16 h), increased the expression of the complex glycosylated form and in parallel the number of hNCC positive cells.
Conclusions. Taken together, the data indicate that 4-phenylbutyrate is a promising candidate for rescuing partially retarded but otherwise functional class II GS mutants.
Keywords: mpk-CCD; NCC; thiazide
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