Enhanced ERK1/2 activity a central feature of cystogenesis in ARPKD. Implications for ion transport phenotype

The purpose of this work was to identify the abnormalities of ion transport in ARPKD cystic cells and to determine the mechanisms that might be responsible for functional abnormalities that lead to fluid accumulation inside cysts. We examined and compared the ion transport phenotype of primary collecting duct cystic and non-cystic monolayers. To that end, 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 non-cystic and cystic mice were selected by fluorescence activated cell sorting (FACS). We evaluated the bioelectrical properties of cystic and non-cystic monolayers and found that Cl^--secretory responses elicited by elevation of cAMP or calcium were not significantly different between normal and cystic monolayers. In contrast, the amiloride-sensitive short-circuit current was significantly reduced in cystic cell monolayers. These studies suggest that cystic cells have an abnormal phenotype reflected by the decrease in ENaC-mediated sodium absorption.

We examined the EGF/EGFR signaling pathway (implicated in pathogenesis of the disease) in vivo. Analysis of cystic kidneys revealed mis-localization of EGF receptor, hyperphosphorylation of EGFR and excessive activation of the p42/44 extracellular signal-regulated protein kinase pathway (ERK1/2) particularly in cyst mural cells. Primary monolayer cultures of non-cystic and cystic collecting duct principal cells were used to determine whether there is a potential role for abnormal EGF/EGFR-dependent regulation of Na⁺ transport in ARPKD. Addition of EGF acutely and chronically to the basolateral bathing solution of non-cystic or cystic monolayers led to p42/44 phosphorylation and inhibition of Na⁺ transport; whereas apical EGF was effective only in monolayers derived from cystic mice. p42/44 phosphorylation and inhibition of Na⁺ transport were prevented by prior treatment of the cells with a MEK inhibitor. Chronic EGF addition to the apical bathing solution of cystic monolayers led to inhibition of Na⁺ transport and decreased ENaC steady state mRNA levels. The results of these studies reveal that the mis-localized apical EGF receptors are functionally coupled to the ERK pathway and that abnormal EGF-dependent ERK1/2-mediated regulation of ENaC function and expression may contribute to PKD pathophysiology.