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1 Rainbow Center for Childhood PKD, Departments of Pediatrics and Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio, USA
* To whom correspondence should be addressed. E-mail: cuc{at}po.cwru.edu.
The main feature of polycystic kidney diseases (PKD) is formation and progressive enlargement of renal cysts. Alterations in epithelial cell proliferation, extracellular matrix, and ion transport are thought to contribute to cyst enlargement and loss of renal function. Abnormal Cl- secretion is implicated in cyst enlargement in autosomal dominant PKD (ADPKD); however, little is known about transport abnormalities associated with autosomal recessive PKD (ARPKD). We developed a method to isolate highly enriched preparations of collecting duct principal cells (site of the lesion in ARPKD) from normal and ARPKD mice. A transgenic mouse (Hoxb7/GFP) in which enhanced green fluorescent protein (GFP) is specifically
expressed in collecting ducts was bred with an ARPKD mouse (BPK), and GFP-positive cells from normal
and cystic mice were selected by fluorescence activated cell sorting (FACS). A large number (1.3±0.2 x 106 cells/mouse; n=32) of highly enriched (>95±3%) GFP-positive collecting duct (CD) cells were obtained from either normal or cystic mice and, when seeded on permeable supports, formed high-resistance (1-1.4 k
cm2 , n=32), polarized epithelial monolayers. Analysis of expression patterns (RT-PCR) for marker proteins, specific for either intercalated cells or principal cells, and the presence of a central cilium confirmed that the monolayers are comprised of principal cells. Under basal conditions, the Cl--secretory responses elicited by elevation of cAMP (
Isc= 2.9±0.3 µA/cm2; n=8 for normal and Isc= 3.2±0.2 µA/cm2; n=8 for cystic) or
calcium (Isc= 13.4±0.8 µA/cm2; n=8 for normal and Isc= 15.3±1.6 µA/cm2; n=7 for cystic) were not significantly different between normal and cystic monolayers. In contrast, the amiloride-sensitive short-circuit current (Isc) was significantly reduced in monolayers of cells isolated from cystic mice (12.9±1.6 µA/cm2; n=10) compared to monolayers of cells isolated from normal mice (27.3±3.4 µA/cm2; n=12). The
results of these studies suggest that ENaC-mediated sodium absorption is decreased in principal cells of ARPKD collecting duct cysts and that the reduction in sodium absorption may contribute to the accumulation
of lumenal fluid.
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