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Basolateral chloride transporters in autosomal dominant polycystic kidney disease
Catherine Lebeau1, Kazushige Hanaoka, Marilyn L Moore-Hoon
1Department of Physiopathology, Université Libre de Bruxelles, Brussels, Belgium.
Insights
In autosomal dominant polycystic kidney disease (ADPKD), cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels drive cyst fluid. The Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) is identified as a key basolateral transporter in most ADPKD cysts.
Area of Science:
- Nephrology
- Molecular Biology
- Cell Biology
Background:
- Transepithelial chloride (Cl(-)) secretion via apical cystic fibrosis transmembrane conductance regulator (CFTR) channels contributes to cyst fluid accumulation in autosomal dominant polycystic kidney disease (ADPKD).
- The basolateral transporters responsible for Cl(-) entry in ADPKD cells remain largely unidentified, though pharmacological evidence implicates a bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporter (NKCC/BSC).
Purpose of the Study:
- To investigate the expression and localization of NKCC1 and anion exchanger type I (AE1) in ADPKD kidneys and cultured ADPKD cells.
- To determine the molecular identity of basolateral Cl(-) transporters involved in ADPKD pathogenesis.
Main Methods:
- Immunoblotting and immunoprecipitation to detect NKCC1 protein.
- Immunostaining of ADPKD kidneys and cultured cells to localize NKCC1, CFTR, and AE1.
- RT-PCR and sequence analysis to confirm gene expression.
Main Results:
- NKCC1 protein was detected in ADPKD cells and kidney extracts.
- Immunostaining revealed basolateral NKCC1 expression in approximately one-third of ADPKD cysts, co-localizing with CFTR.
- AE1 was found in CFTR-positive cysts lacking NKCC1, suggesting an alternative pathway.
Conclusions:
- The findings indicate that NKCC1 is a major basolateral Cl(-) transporter in CFTR-positive ADPKD cysts, similar to secretory epithelia.
- AE1 may serve as an alternative basolateral Cl(-) pathway in a subset of ADPKD cysts.
Abstract:
Transepithelial Cl(-) secretion mediated by apical cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels plays a key role in cyst fluid accumulation in autosomal dominant polycystic kidney disease (ADPKD). The molecular identity of the basolateral transporter(s) responsible for Cl(-) entry in ADPKD cells is unknown, although pharmacological studies suggest that a bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporter (NKCC/BSC) is involved. We investigated the expression of NKCC1, CFTR and anion exchanger type I (AE1) in ADPKD kidneys and cultured ADPKD cells. Immunoblotting and immunoprecipitation detected NKCC1 at ~170 kDa in ADPKD cells and kidney extracts. Immunostaining located NKCC1 in one-third of ADPKD cysts, with a pattern of basolateral reactivity. Staining of serial sections showed that cysts positive for NKCC1 also stained for CFTR. Additional studies demonstrated that AE1 is expressed in ADPKD kidneys, and is located at the basolateral pole of CFTR-positive ADPKD cysts that do not express NKCC1. RT-PCR and sequence analyses confirmed the selective expression of NKCC1 or AEI in cultured ADPKD cells that also express CFTR. The fact that most CFTR-positive ADPKD cysts also express NKCC1 suggests that transepithelial Cl(-) secretion in ADPKD involves molecular mechanisms similar to secretory epithelia. AE1 might be an alternative basolateral pathway for Cl(-) in a minority of cysts.